Sample records for a-deficient apre mutant

We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mumol/m2/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

ABSTRACT Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. PMID:27601574

Understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, β-glucan, or fructan, such as functional foods or oils for biofuel applications. Waxy mutants of cereals have a high content of amylopectin and have been well characterized. However, the allocation of carbon to other components, such as β-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. In this study, we used a rice mutant, GM077, with a low content of amylose to gain molecular insight into how adeficiency of amylose affects carbon allocation to other end products and to amylopectin. We used carbohydrate analysis, subtractive cDNA libraries, and qPCR to identify candidate genes potentially responsible for the changes in carbon allocation in GM077 seeds. Carbohydrate analysis indicated that the content of amylose in GM077 seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type BP034. The content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. Suppression subtractive hybridization (SSH) experiments generated 116 unigenes in the mutant on the wild-type background. Among the 116 unigenes, three, AGP, ISA1 and SUSIBA2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. A bioinformatics analysis of the putative SUSIBA2-like binding elements in the promoter regions of the upregulated genes indicated that the SUSIBA2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. Analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant GM077

Background Understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, β-glucan, or fructan, such as functional foods or oils for biofuel applications. Waxy mutants of cereals have a high content of amylopectin and have been well characterized. However, the allocation of carbon to other components, such as β-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. In this study, we used a rice mutant, GM077, with a low content of amylose to gain molecular insight into how adeficiency of amylose affects carbon allocation to other end products and to amylopectin. We used carbohydrate analysis, subtractive cDNA libraries, and qPCR to identify candidate genes potentially responsible for the changes in carbon allocation in GM077 seeds. Results Carbohydrate analysis indicated that the content of amylose in GM077 seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type BP034. The content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. Suppression subtractive hybridization (SSH) experiments generated 116 unigenes in the mutant on the wild-type background. Among the 116 unigenes, three, AGP, ISA1 and SUSIBA2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. A bioinformatics analysis of the putative SUSIBA2-like binding elements in the promoter regions of the upregulated genes indicated that the SUSIBA2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. Conclusion Analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (kcat/Km) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45°C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176(th) residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca(2+), which increased the thermostability of M179.

Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-γ-glutamate (γ-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in γ-PGA production and in the total extracellular protease activity. The defect in γ-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline serine protease encoded by aprE plays an indispensable role in supplying materials to produce γ-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either γ-PGA production or total protease activity.

In high copy number, the Escherichia coli mprA gene reduces the synthesis of peptide microcins B17 and C7 (MccB17 and MccC7) and blocks the osmoinduction of the proU operon at the transcriptional level. mprA has been sequenced and shown to encode a polypeptide of 176 amino acids (Mr, 20,563). Insertion and deletion mutant mprA alleles were constructed and then transferred to the chromosome by allelic replacement. In these mutants, expression of two mcb-lacZ fusions was fivefold derepressed, indicating a negative regulatory role of mprA on the mcb operon (MccB17). In contrast, no effect of the MprA- mutations on the expression of mcc operon (MccC7) or on the osmoinduction of proU operon was observed. PMID:1840583

Characterization of a copA(-) mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA(-) mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg-chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe-4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.

The Second Generation Airborne Precipitation Radar (APR-2) participated in the Genesis and Rapid Intensification Processes (GRIP) experiment in August and September of 2010, collecting a large volume of data in several tropical systems, including Hurricanes Earl and Karl. Additional measurements of tropical cyclone have been made by APR-2 in experiments prior to GRIP (namely, CAMEX-4, NAMMA, TC4); Table 1 lists all the APR-2 tropical cyclone observations. The APR-2 observations consist of the vertical structure of rain reflectivity at 13.4 and 35.6 GHz, and at both co-polarization and crosspolarization, as well as vertical Doppler measurements and crosswind measurements. APR-2 normally flies on the NASA DC-8 aircraft, as in GRIP, collecting data with a downward looking, cross-track scanning geometry. The scan limits are 25 degrees on either side of the aircraft, resulting in a roughly 10-km swath, depending on the aircraft altitude. Details of the APR-2 observation geometry and performance can be found in Sadowy et al. (2003).The multiparameter nature of the APR-2 measurements makes the collection of tropical cyclone measurements valuable for detailed studies of the processes, microphysics and dynamics of tropical cyclones, as well as weaker systems that are associated with tropical cyclone formation. In this paper, we give a brief overview of how the APR-2 data are processed. We also discuss use of the APR-2 cross-track winds to estimate various quantities of interest in in studies of storm intensification. Finally, we show examples of the standard products and derived information.

The Second Generation Airborne Precipitation Radar (APR-2) participated in the Genesis and Rapid Intensification Processes (GRIP) experiment in August and September of 2010, collecting a large volume of data in several tropical systems, including Hurricanes Earl and Karl. Additional measurements of tropical cyclone have been made by APR-2 in experiments prior to GRIP (namely, CAMEX-4, NAMMA, TC4); Table 1 lists all the APR-2 tropical cyclone observations. The APR-2 observations consist of the vertical structure of rain reflectivity at 13.4 and 35.6 GHz, and at both co-polarization and crosspolarization, as well as vertical Doppler measurements and crosswind measurements. APR-2 normally flies on the NASA DC-8 aircraft, as in GRIP, collecting data with a downward looking, cross-track scanning geometry. The scan limits are 25 degrees on either side of the aircraft, resulting in a roughly 10-km swath, depending on the aircraft altitude. Details of the APR-2 observation geometry and performance can be found in Sadowy et al. (2003).The multiparameter nature of the APR-2 measurements makes the collection of tropical cyclone measurements valuable for detailed studies of the processes, microphysics and dynamics of tropical cyclones, as well as weaker systems that are associated with tropical cyclone formation. In this paper, we give a brief overview of how the APR-2 data are processed. We also discuss use of the APR-2 cross-track winds to estimate various quantities of interest in in studies of storm intensification. Finally, we show examples of the standard products and derived information.

Respirators: APR Issuer Self-Study (course 33461) is designed to introduce and familiarize employees selected as air-purifying respirator (APR) issuers at Los Alamos National Laboratory (LANL) with the responsibilities, limitations, procedures, and resources for issuing APRs at LANL. The goal is to enable these issuers to consistently provide proper, functioning APRs to authorized users

Immunoglobulin class A is the main protein of the mucosal immune system. Selective immunoglobulin Adeficiency (sIgAD) is the most common primary immunodeficiency in Caucasians. sIGAD is strongly associated with the certain major histocompatibility complex region. Most individuals with sIgAD are asymptomatic and identified coincidentally. However, some patients may present with recurrent infections, allergic disorders and autoimmune manifestations. Several autoimmune diseases, such as systemic lupus erythematosus, diabetes mellitus type 1, Graves disease and celiac disease, are associated with an increased prevalence of sIgAD. Screening for sIgAD in coeliac disease is essential. Patients need treatment of associated diseases. It is also known that IgAdeficiency may progress into a common variable immunodeficiency (CVID). Pathogenesis and molecular mechanism involved in sIgAD should be elucidated in the future.

Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

d-Serine, an N-methyl d-aspartate receptor coagonist, and its regulatory enzymes, d-amino acid oxidase (DAO; degradation) and serine racemase (SR; synthesis), have been implicated in crucial roles of the developing central nervous system, yet the functional position that they play in regulating the availability of d-serine throughout development of the mammalian retina is not well-known. Using capillary electrophoresis and a sensitive method of enantiomeric amino acid separation, we were able to determine total levels of d-serine at specific ages during postnatal development of the mouse retina in two different strains of mice, one of which contained a loss-of-function point mutation for DAO while the other was a SR knockout line. Each mouse line was tested against conspecific wild type (WT) mice for each genetic strain. The universal trend in all WT and transgenic mice was a large amount of total retinal d-serine at postnatal age 2 (P2), followed by a dramatic decrease as the mice matured into adulthood (P70–80). SR knockout mice retinas had 41% less d-serine than WT retinas at P2, and 10 times less as an adult. DAO mutant mice retinas had significantly elevated levels of d-serine when compared to WT retinas at P2 (217%), P4 (223%), P8 (194%), and adulthood (227%). PMID:25083578

This document is intended to assist State Education Agency (SEA) and Lead Agency (LA) staff and technical assistance providers in designing a meaningful evaluation for the State Performance Plan (SPP)/Annual Performance Report (APR) improvement activities. It provides: (1) information about the relevance of evaluation in the context of improvement…

Na-APR-1(M74) is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1(M74) and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1 wt in vitro, thereby providing proof of concept of Na-APR-1(M74) as a vaccine antigen. The mutated version, Na-APR-1(M74), was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1(M74) IgG. As little as 0.99 μg of recombinant Na-APR-1(M74) could induce anti Na-APR-1(M74) IgG in mice that were capable of inhibiting Na-APR-1w t-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1(M74) as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1 wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1(M74) which was assessed by fluorescence polarization, but with an ∼ 50-fold reduction in the dissociation constant. Taken together, these assays comprise a "toolbox" that could be useful for the analyses of Na-APR-1(M74) as it

Na-APR-1M74 is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1M74 and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1wt in vitro, thereby providing proof of concept of Na-APR-1M74 as a vaccine antigen. The mutated version, Na-APR-1M74, was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1M74 IgG. As little as 0.99 μg of recombinant Na-APR-1M74 could induce anti Na-APR-1M74 IgG in mice that were capable of inhibiting Na-APR-1wt-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1M74 as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1M74 which was assessed by fluorescence polarization, but with an ∼50-fold reduction in the dissociation constant. Taken together, these assays comprise a “toolbox” that could be useful for the analyses of Na-APR-1M74 as it proceeds through the

Background One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. Methods The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)–labeled casein-substrate. Results The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. Conclusion It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase. PMID:25215183

OBJECTIVE: To review cases of vitamin Adeficiency and the effects of vitamin A supplementation on child morbidity and mortality. METHODS: Articles published in scientific journals, technical and scientific books, and also publications by international organizations were used as source of information. RESULTS: Clinical manifestations of xerophthalmia affect the retina (night blindness), the conjunctiva (conjunctival xerosis, with or without Bitot spots), and the cornea (corneal xerosis). Corneal xerosis may lead to corneal ulceration and liquefactive necrosis (keratomalacia). A priori, these signs and symptoms are the best indicators of vitamin Adeficiency; they are, however, extremely rare. Laboratory indicators include Conjunctival Impression Cytology and serum retinol concentrations. The World Health Organization (WHO) recommends the use of two biological markers in order to characterize vitamin Adeficiency in a given population. If only one biological marker is used, this marker has to be backed up by a set of at least four additional risk factors. Corneal xerophthalmia should be treated as a medical emergency; In the event of suspected vitamin Adeficiency, a 200,000 IU vitamin A dose should be administered orally, repeating the dose after 24 hours (half the dose for infants younger than one year). Vitamin A supplementation in endemic areas may cause a 23 to 30% reduction in the mortality rate of children aged between 6 months and five years, and attenuate the severity of diarrhea. The methods for the control of vitamin Adeficiency are available in the short (supplementation with megadoses), medium (food fortification), and long run (diet diversification). CONCLUSION: There is evidence of vitamin Adeficiency among Brazilian children. Pediatricians must be aware of the signs and symptoms of this disease, however sporadic they might be. It is of paramount importance that vitamin A be included in public policy plans so that we can ensure the survival of

IgAdeficiency is the most common primary immunodeficiency. Early diagnosis and clinical follow-up may improve the quality of life of patients with IgAdeficiency. To this end, IgAdeficiency should be further studied and better understood on its clinical manifestations. To determine IgAdeficiency clinical manifestations. Cross-sectional, retrospective, exploratory study, where the medical records of 39 patients with IgAdeficiency were analyzed. Among the analyzed cases, 10 patients were diagnosed with total IgAdeficiency and 29 patients with partial IgAdeficiency. Partial and total IgAdeficiency main clinical manifestations were allergic rhinoconjunctivitis and allergic asthma. In total IgAdeficiency, in addition to allergic diseases, a statistically significant number (p < 0.05) of cases of infection-related rhinosinusitis, tonsillitis and conjunctivitis were also observed. This study showed that the main clinical manifestations in IgAdeficiency were allergic rhinoconjunctivitis and allergic asthma. In addition, patients with total IgAdeficiency showed a significant increase in infection-related rhinosinusitis, tonsillitis and conjunctivitis, when compared with patients with partial IgAdeficiency.

Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre-) peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment. PMID:23112874

Designing Air Cleaning Units (ACU) of an Engineered Safety Feature and normal atmosphere clean-up system at the renovated APR-1000 and APR-1400 NPP, and fuel cycle facilities in Korea, is required to meet the standards of ASME AG-1 (1997), ASME N509/N510 (1989) and KEPIC-MH (2001) to enhance the removal efficiency of aerosols and particulates from the effluents. The revised ACU testing criteria are allowed to use alternative challenge agents of the dioctyl phthalate and Refrigerant-11 for in situ testing of high efficiency particulate air filters and adsorption banks. The operability testing time of engineered safety feature (ESF) trains was changed from 10 h to 15 min. The activated carbon in adsorption banks should undergo laboratory tests at a temperature of 30 °C and relative humidity 95 %. The removal criteria of methyl iodide should be over 99.5 % for ESF and 99 % for normal systems. This paper provides the background of the changed criteria for designing and testing of the ACU system in nuclear facilities.

... Regulations or APR implement certain obligations of the United States under the Protocol Additional to the Agreement Between the United States of America and the International Atomic Energy Agency Concerning the Application of Safeguards in the United States of America, known as the Additional Protocol. (a) Persons and...

Analysis of arylsulfatase A from pseudo arylsulfatase Adeficiency fibroblasts by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoradiochemical nitrocellulose blot radiography revealed two subunit bands which migrated faster than subunit bands of enzyme from normal fibroblasts. Immunoreactive material was present only at levels comparable to enzyme activity. These findings imply that arylsulfatase A in pseudodeficiency is structurally altered, but it is catalytically equivalent to normal arylsulfatase A. This altered enzyme must be the product of the pseudodeficiency gene since no immunoreactive product of the metachromatic leukodystrophy gene could be detected in metachromatic leukodystrophy cells by the procedure employed. It is not clear from the present data if the attenuated arylsulfatase A activity in pseudodeficiency results from a decreased rate of synthesis or an increased lability of the mutant enzyme.

The vitamin A-deficient keratinized cornea is very susceptible to ulceration possibly due to altered stromal components. In this study the proteoglycans present in the corneal stroma of vitamin A-deficient, pair-fed and normal rabbits were compared. Rabbits after weaning were placed on a vitamin Adeficient diet, the same diet with retinyl palmitate added (pair-fed) or normal rabbit chow. After 5 months, the corneas of the vitamin A-deficient animals became keratinized. The corneal components were then labeled by injection of /sup 3/H-leucine and Na/sup 35/SO/sub 4/ into the anterior chamber of the eyes on 3 successive days. On the 4th day the animals were sacrificed the corneas removed and dissected. The labeled corneal stromas were extracted with 4 M GuHCl and the components separated on a DEAE-Sepharose column. The proteoglycans were eluted with 0.5 M and 1.0 M NaCl. The 1.0 M NaCl fraction (mainly keratin sulfate proteoglycans) was increased 25% in the vitamin A-deficient corneas over that for the pair-fed and normal corneas. These proteoglycans from the deficient corneas gave a different elution pattern on Octyl-Sepharose eluted with a Triton X-100 gradient than those from the pair-fed corneas. The total labeled proteoglycans were similar in the stromas from the 3 types of rabbits. These results indicate the various corneal proteoglycan ratios differ under vitamin Adeficiency conditions.

Due to the rapid advancement of digital technology, the definite technical advantages of digital control system compared to analog control system are accelerating the implementation of advanced distributed digital control system in the nuclear power plant. One of the major advantages of digital control system is the capability of Soft Control System. The design of Soft Control System for Advanced Power Reactor 1400 (APR1400) plant of Man-Machine Interface System (MMIS) is based on full digital technologies to enhance reliability, operability and maintainability. Computer-based compact workstation has been adopted in the APR1400 Main Control Room (MCR) to provide convenient working environment. This paper introduces the approaches and methodologies of Soft Control System for the Advanced Control Room (ACR). This paper also explains major design features for operation and display of the Soft Control System and its implementation to cope with regulatory requirements. (authors)

Low mean concentrations of serum immunoglobulin A (IgA) and an increased frequency of overt IgAdeficiency (IgAD) in certain dog breeds raises the question whether it is a breeding-enriched phenomenon or a legacy from the dog's ancestor, the gray wolf (Canis lupus). The IgA concentration in 99 serum samples from 58 free-ranging and 13 captive Scandinavian wolves, was therefore measured by capture ELISA. The concentrations were markedly lower in the wolf serum samples than in the dog controls. Potential differences in the IgA molecule between dogs and wolves were addressed by sequencing the wolf IgA heavy chain constant region encoding gene (IGHA). Complete amino acid sequence homology was found. Detection of wolf and dog IgA was ascertained by showing identity using double immunodiffusion. We suggest that the vast majority of wolves, the ancestor of the dog, are IgAdeficient.

Brookhaven National Laboratory (BNL) assisted the Korea Institute of Nuclear Safety (KINS) in reviewing the Level 2 Probabilistic Safety Assessment (PSA) of the APR+ Advanced Pressurized Water Reactor (PWR) prepared by the Korea Hydro & Nuclear Power Co., Ltd (KHNP) and KEPCO Engineering & Construction Co., Inc. (KEPCO-E&C). The work described in this report involves a review of the APR+ Level 2 PSA submittal [Ref. 1]. The PSA and, therefore, the review is limited to consideration of accidents initiated by internal events. As part of the review process, the review team also developed three sets of Requests for Additional Information (RAIs). These RAIs were provided to KHNP and KEPCO-E&C for their evaluation and response. This final detailed report documents the review findings for each technical element of the PSA and includes consideration of all of the RAIs made by the reviewers as well as the associated responses. This final report was preceded by an interim report [Ref. 2] that focused on identifying important issues regarding the PSA. In addition, a final meeting on the project was held at BNL on November 21-22, 2011, where BNL and KINS reviewers discussed their preliminary review findings with KHNP and KEPCO-E&C staffs. Additional information obtained during this final meeting was also used to inform the review findings of this final report. The review focused not only on the robustness of the APR+ design to withstand severe accidents, but also on the capability and acceptability of the Level 2 PSA in terms of level of detail and completeness. The Korean nuclear regulatory authorities will decide whether the PSA is acceptable and the BNL review team is providing its comments for KINS consideration. Section 2.0 provides the basis for the BNL review. Section 3.0 presents the review of each technical element of the PSA. Conclusions and a summary are presented in Section 4.0. Section 5.0 contains the references.

A 50-year-old woman developed pronounced IgA-deficiency and hypothyroidism after 131I treatment for Graves' disease. The deficiency state was associated with a severe sinobronchial syndrome. Treatment with L-thyroxine resulted in a normal IgA concentration and a dramatic clinical improvement. Of the various possible underlying mechanisms, impaired synthesis of IgA light and heavy light chains seemed most probable. Impaired production of J-chain was excluded. PMID:7339604

ARID1A, a chromatin remodeler of the SWI/SNF family, is a recently identified tumor suppressor that is mutated in a broad spectrum of human cancers. Thus, it is of fundamental clinical importance to understand its molecular functions and determine whether ARID1Adeficiency can be exploited therapeutically. In this manuscript, we report a key function of ARID1A in regulating the DNA damage checkpoint. ARID1A is recruited to DNA double strand breaks (DSBs) via its interaction with the upstream DNA damage checkpoint kinase ATR. At the molecular level, ARID1A facilitates efficient processing of DSB to single strand ends, and sustains DNA damage signaling. Importantly, ARID1Adeficiency sensitizes cancer cells to PARP inhibitors in vitro and in vivo providing a potential therapeutic strategy for patients with ARID1A-mutant tumors. PMID:26069190

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

APR 1400 is a PWR (Pressurized Water Reactor) with rated power of 3983 MWth and 241 assemblies. Recently, demand for extremely longer cycle up to 24 months is increasing with challenge of higher critical boron concentration (CBC). In this paper, assembly design method of selecting Gd-rods is introduced to reduce CBC. The purpose of the method is to lower the critical boron concentration of the preliminary core loading pattern (PLP), and consequently to achieve more negative or less positive moderator temperature coefficient (MTC). In this method, both the ratio of the number of low-Gd rod to the number of high-Gd rod (r) and assembly average Gd wt% (w) are the decision variables. The target function is the amount of soluble boron concentration reduction, which can be converted to ΔkTARGET. A set of new designed fuel assembly satisfies an objective function, min [f =∑i (ΔkF A-Δki ) ] , and enables a final loading pattern to reach a target CBC. The constraints required to determine a set of Δk are physically realizable pair, (r,w), and the sum of Δk of new designed assemblies as close to ΔkTARGET as possible. New Gd-bearing assemblies selected based on valid pairs of (r,w) are replaced with existing assemblies in a PLP. This design methodology is applied to Shin-Kori Unit 3 Cycle 1 used as a reference model. CASMO-3/MASTER code is used for depletion calculation. CASMO-3/MASTER calculations with new designed assemblies produce lower CBC than the expected CBC, proving that the proposed method works successful.

APR 1400 is a PWR (Pressurized Water Reactor) with rated power of 3983 MWth and 241 assemblies. Recently, demand for extremely longer cycle up to 24 months is increasing with challenge of higher critical boron concentration (CBC). In this paper, assembly design method of selecting Gd-rods is introduced to reduce CBC. The purpose of the method is to lower the critical boron concentration of the preliminary core loading pattern (PLP), and consequently to achieve more negative or less positive moderator temperature coefficient (MTC). In this method, both the ratio of the number of low-Gd rod to the number of high-Gd rod (r) and assembly average Gd wt% (w) are the decision variables. The target function is the amount of soluble boron concentration reduction, which can be converted to Δk{sub TARGET}. A set of new designed fuel assembly satisfies an objective function, min [f=∑{sub i}(Δk{sub FA}−Δk{sub i})], and enables a final loading pattern to reach a target CBC. The constraints required to determine a set of Δk are physically realizable pair, (r,w), and the sum of Δk of new designed assemblies as close to Δk{sub TARGET} as possible. New Gd-bearing assemblies selected based on valid pairs of (r,w) are replaced with existing assemblies in a PLP. This design methodology is applied to Shin-Kori Unit 3 Cycle 1 used as a reference model. CASMO-3/MASTER code is used for depletion calculation. CASMO-3/MASTER calculations with new designed assemblies produce lower CBC than the expected CBC, proving that the proposed method works successful.

...)(APR 1996) (a) The Project Officer (PO) or his/her designee, after a written determination by the... General Counsel for Information Law or his/her designee; and (iii) return the CBI to the PO or...

... Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL... (see § 782.5 of the APR). In addition, forms may be downloaded from the Internet at http://www.ap.gov....

... (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL REGULATIONS GENERAL... forms required by the APR may be downloaded from the Internet at http://www.ap.gov. You also may...

... Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL... (see § 782.5 of the APR). In addition, forms may be downloaded from the Internet at http://www.ap.gov....

... (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL REGULATIONS GENERAL... forms required by the APR may be downloaded from the Internet at http://www.ap.gov. You also may...

... (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL REGULATIONS GENERAL... forms required by the APR may be downloaded from the Internet at http://www.ap.gov. You also may...

... (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL REGULATIONS GENERAL... forms required by the APR may be downloaded from the Internet at http://www.ap.gov. You also may...

... Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL... (see § 782.5 of the APR). In addition, forms may be downloaded from the Internet at http://www.ap.gov....

... Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL... (see § 782.5 of the APR). In addition, forms may be downloaded from the Internet at http://www.ap.gov....

... Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL... (see § 782.5 of the APR). In addition, forms may be downloaded from the Internet at http://www.ap.gov....

... (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE ADDITIONAL PROTOCOL REGULATIONS GENERAL... forms required by the APR may be downloaded from the Internet at http://www.ap.gov. You also may...

Vitamin Adeficiency may occur after malabsorptive bariatric surgery. However, it rarely entails important functionally limiting symptoms. We present the case of a woman who underwent bariatric surgery and developed ocular dryness, xeroderma and hearing loss due to severe vitamin Adeficiency. We illustrate an outstanding and exceptional case of the consequences of an excessive and uncontrolled malabsorption.

The Advanced Power Reactor Plus (APR +) nuclear power plant design has been developed on the basis of the APR1400 (Advanced Power Reactor 1400 MWe) to further enhance safety and economics. For the mitigation of Anticipated Transients Without Scram (ATWS) as well as Common Cause Failures (CCF) within the Plant Protection System (PPS) and the Emergency Safety Feature - Component Control System (ESF-CCS), several design improvement features have been implemented for the Diverse Protection System (DPS) of the APR + plant. As compared to the APR1400 DPS design, the APR + DPS has been designed to provide the Safety Injection Actuation Signal (SIAS) considering a large break LOCA accident concurrent with the CCF. Additionally several design improvement features, such as channel structure with redundant processing modules, and changes of system communication methods and auto-system test methods, are introduced to enhance the functional reliability of the DPS. Therefore, it is expected that the APR + DPS can provide an enhanced safety and reliability regarding possible CCF in the safety-grade I and C systems as well as the DPS itself. (authors)

This paper presents an automated tool for optimizing the routing of primary cables in underground residential distribution systems (URD). Although initially developed as one of the modules in Puget Sound Power and Light Co.`s automated electrical plant design program, Automated Primary Router (APR) can also be used in standalone mode. APR, implemented in a Geographic Information System (GIS)-based environment, is capable of efficiently accessing and manipulating geographically referenced data from a facilities management system database. It also provides a full-fledged Graphic User Interface, along with on-line visualization and accurate cost estimation to facilitate the design process. APR employs a heuristic search algorithm to find the best primary cable routes either for new residential developments, or for existing URD systems as part of the ongoing cable replacement program. Based on the test results, APR shows significant stability and efficiency in finding the optimal solution for primary cable routing. With this performance, APR can help distribution engineers improve the quality of URD design, producing standardized and economically justifiable primary cable routes. Because geographically referenced design data are digitally stored in the GIS database, APR further increases the reusability and accessibility of the URD design information.

This paper presents an automated tool for optimizing the routing of primary cables in underground residential distribution systems (URD). Although initially developed as on of the modules in Puget Sound Power and Light Co.`s automated electrical plant design program, Automated Primary Router (APR) can also be used in standalone mode. APR, implemented in a Geographic Information System (GIS)-based environment, is capable of efficiently accessing and manipulating geographically referenced data from a facilities management system database. It also provides a full-fledged Graphic User Interface, along with on-line visualization and accurate cost estimation to facilitate the design process. APR employs a heuristic search algorithm to find the best primary cable routes whether for new residential developments, or for existing URD systems as part of the ongoing cable replacement program. Based on the test results, APR shows significant stability and efficiency in finding the optimal solution for primary cable routing. With this performance, APR can help distribution engineers improve the quality of URD design, producing standardized and economically justifiable primary cable routes. Because geographically referenced design data are digitally stored in the GIS database, APR further increases the reusability and accessibility of the URD design information.

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC(-), through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC(-) inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC(-) antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11-glutathione axis.

TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC−, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC− inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC− antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis. PMID:28348409

Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

We have investigated the consequences of vitamin Adeficiency in a rat model of T-cell-dependent and superantigen-mediated Staphylococcus aureus arthritis. After intravenous inoculation of enterotoxin A-producing staphylococci, the vitamin-A-deficient rats showed a decreased weight gain compared with the paired fed controls despite equal food consumption. The control rats developed arthritis in the first few days after bacterial inoculation, with a peak frequency at day 5, and then gradually recovered; however, the frequency of arthritis 18 days after bacterial inoculation was 86% among the vitamin A-deficient rats and 44% among the control rats. During this period, 3 of 10 deficient rats and 1 of 10 control rats died. Further in vitro analysis revealed that T-cell responses to S. aureus were significantly higher in the vitamin A-deficient rats than in the control animals. In contrast, B-cell reactivity, measured as immunoglobulin levels, autoantibody levels, and specific antibacterial antibody levels in serum, did not differ between the groups. Interestingly, the innate host defense mechanisms against S. aureus were also profoundly affected by vitamin Adeficiency. Thus, despite a larger number of circulating phagocytic cells in the vitamin-A-deficient group, the capacity to phagocytize and exert intracellular killing of S. aureus was significantly decreased in comparison with the control rats. Furthermore, serum from the vitamin A-deficient rats inoculated with Staphylococcus aureus displayed decreased complement lysis activity. Our results suggest that the increased susceptibility to S. aureus infection observed in the vitamin-A-deficient rats is due to a concerted action of antigen-specific T-cell hyperactivity, impaired function of the phagocytes, and decreased complement activity. PMID:8557341

Up to now only 3 cases of extrinsic allergic alveolitis (hypersensitivity pneumonitis) with IgAdeficiency have been published worldwide. We had the opportunity to detect two additional cases which will be presented here. Summarizing all cases IgAdeficiency is a risk factor for a severe course of the disease and an increased susceptibility to acquire allergic alveolitis by low dose antigen exposure.

APR1400 is an evolutionary PWR developed in Korea. The ECCS of APR1400 has been improved by adopting 4 safety injection pumps and fluidic devices in safety injection tanks. Also, APR1400 adopted direct vessel injection (DVI) for its ECCS injection: safety injection water is injected into the reactor vessel downcomer directly. The ECCS performance of APR1400 during the double-ended cold leg break LOCA, specially during the late reflood phase, is examined with the KEPRI Realistic Evaluation Methodologies (KREM). The applicability of KREM to the prediction of APR1400 specific thermal hydraulic phenomena in downcomer during the LBLOCA is examined first and the preliminary best estimate analysis is performed for the double-ended cold leg break LOCA. The analysis results show that there is no degradation of core coolability and reheating phenomena during the late reflood phase. All fuel rods are quenched in the early reflood phase during the fluidic devices are active, which shows the effectiveness of the fluidic devices in mitigating the LBLOCA. (authors)

This paper presents the technical description, mechanical design, electronic components, software implementation and possible applications of a tele-operated mobile robot designed as an assisted living tool. This robotic concept has been named Assistant Personal Robot (or APR for short) and has been designed as a remotely telecontrolled robotic platform built to provide social and assistive services to elderly people and those with impaired mobility. The APR features a fast high-mobility motion system adapted for tele-operation in plain indoor areas, which incorporates a high-priority collision avoidance procedure. This paper presents the mechanical architecture, electrical fundaments and software implementation required in order to develop the main functionalities of an assistive robot. The APR uses a tablet in order to implement the basic peer-to-peer videoconference and tele-operation control combined with a tactile graphic user interface. The paper also presents the development of some applications proposed in the framework of an assisted living robot.

OBJECTIVE: To determine if a widely used proprietary risk-adjustment system, APR-DRGs, misadjusts for severity of illness and misclassifies provider performance. DATA SOURCES: (1) Discharge abstracts for 116,174 noninstitutionalized adults with acute myocardial infarction (AMI) admitted to nonfederal California hospitals in 1991-1993; (2) inpatient medical records for a stratified probability sample of 974 patients with AMIs admitted to 30 California hospitals between July 31, 1990 and May 31, 1991. STUDY DESIGN: Using the 1991-1993 data set, we evaluated the predictive performance of APR-DRGs Version 12. Using the 1990/1991 validation sample, we assessed the effect of assigning APR-DRGs based on different sources of ICD-9-CM data. DATA COLLECTION/EXTRACTION METHODS: Trained, blinded coders reabstracted all ICD-9-CM diagnoses and procedures, and established the timing of each diagnosis. APR-DRG Risk of Mortality and Severity of Illness classes were assigned based on (1) all hospital-reported diagnoses, (2) all reabstracted diagnoses, and (3) reabstracted diagnoses present at admission. The outcome variables were 30-day mortality in the 1991-1993 data set and 30-day inpatient mortality in the 1990/1991 validation sample. PRINCIPAL FINDINGS: The APR-DRG Risk of Mortality class was a strong predictor of death (c = .831-.847), but was further enhanced by adding age and sex. Reabstracting diagnoses improved the apparent performance of APR-DRGs (c = .93 versus c = .87), while using only the diagnoses present at admission decreased apparent performance (c = .74). Reabstracting diagnoses had less effect on hospitals' expected mortality rates (r = .83-.85) than using diagnoses present at admission instead of all reabstracted diagnoses (r = .72-.77). There was fair agreement in classifying hospital performance based on these three sets of diagnostic data (K = 0.35-0.38). CONCUSIONS: The APR-DRG Risk of Mortality system is a powerful risk-adjustment tool, largely because it

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions.

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions. PMID:26502908

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.

Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

... Regulatory Commission. Nuclear fuel cycle-related research and development. Those activities that are...) of the APR for nuclear fuel cycle-related activities subject to these reporting requirements). Reporting Code. A unique identification used for identifying a location where one or more nuclear fuel cycle...

... Regulatory Commission. Nuclear fuel cycle-related research and development. Those activities that are...) of the APR for nuclear fuel cycle-related activities subject to these reporting requirements). Reporting Code. A unique identification used for identifying a location where one or more nuclear fuel cycle...

... Regulatory Commission. Nuclear fuel cycle-related research and development. Those activities that are...) of the APR for nuclear fuel cycle-related activities subject to these reporting requirements). Reporting Code. A unique identification used for identifying a location where one or more nuclear fuel cycle...

... Regulatory Commission. Nuclear fuel cycle-related research and development. Those activities that are...) of the APR for nuclear fuel cycle-related activities subject to these reporting requirements). Reporting Code. A unique identification used for identifying a location where one or more nuclear fuel cycle...

... Regulatory Commission. Nuclear fuel cycle-related research and development. Those activities that are...) of the APR for nuclear fuel cycle-related activities subject to these reporting requirements). Reporting Code. A unique identification used for identifying a location where one or more nuclear fuel cycle...

Participation in rigorous high school courses such as Advanced Placement (AP[R]) Physics increases the likelihood of college success, especially for students who are traditionally underserved. Tackling difficult multiple-choice exams should be part of any AP program because well-constructed multiple-choice questions, such as those on AP exams and…

Advanced Placement[R] (AP[R]) courses provide high school students an opportunity to be exposed to the rigors of college while they are still in high school. Since school policies play a role in the opportunities for students to pursue these courses, educational leaders must be aware of the ways in which their personal beliefs and school practices…

... failure, transmit signals, actuate fuel controls or power levers on operating engines, including power... power lever position; and (c) That shown to be free of hazardous engine response characteristics when...; (3) Prevent deactivation of the APR by manual adjustment of the power levers following an engine...

... failure, transmit signals, actuate fuel controls or power levers on operating engines, including power... power lever position; and (c) That shown to be free of hazardous engine response characteristics when...; (3) Prevent deactivation of the APR by manual adjustment of the power levers following an engine...

... failure, transmit signals, actuate fuel controls or power levers on operating engines, including power... power lever position; and (c) That shown to be free of hazardous engine response characteristics when...; (3) Prevent deactivation of the APR by manual adjustment of the power levers following an engine...

... failure, transmit signals, actuate fuel controls or power levers on operating engines, including power... power lever position; and (c) That shown to be free of hazardous engine response characteristics when...; (3) Prevent deactivation of the APR by manual adjustment of the power levers following an engine...

... failure, transmit signals, actuate fuel controls or power levers on operating engines, including power... power lever position; and (c) That shown to be free of hazardous engine response characteristics when...; (3) Prevent deactivation of the APR by manual adjustment of the power levers following an engine...

Vitamin Adeficiency is a rare but vision threatening disorder in the developed world, which can lead to blindness for severe keratomalacia with cornea scarring and perforation or night blindness due to impaired dark adaptation. Conversely, the disease is quite common in developing countries, as a consequence of chronic malnutrition. The correct diagnosis and therapy with prompt vitamin A supplementation avoid blindness. We report a series of 3 local cases with different age and causes for vitamin Adeficiency. The diagnostic workup, therapy, and prognosis are discussed. PMID:26509090

Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs' essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.

Oral tolerance is a phenomenon of induction of systemic unresponsiveness to antigens ingested by the oral route and loss of immune response. Studies have shown the importance of vitamin A in oral tolerance in vitro but not in an in vivo experimental model. Therefore, we carried out experiments to determine how vitamin Adeficiency affects tolerance induction and the ability of mesenteric lymph node (MLN) CD11c(+) cells to induce regulatory T cells (Tregs). Immunological tolerance was induced by oral ovalbumin (OVA) administration in vitamin A-sufficient mice. OVA-specific antibody and cytokine production were significantly reduced. On the other hand, in vitamin A-deficient mice, both OVA-specific antibody and cytokine production were not suppressed by oral OVA administration. Regarding induction of Tregs, the conversion rate of Foxp3(+) cells from naïve CD4(+) cell by CD11c(+) cells was decreased in vitamin A-deficient mice. Our study indicates that vitamin Adeficiency causes the breakdown of oral tolerance in vivo.

California is a major producer of tangerines and oranges, which contain carotenoids that form vitamin A. Deficiencies of this vitamin are common in southern Asia and Africa, causing blindness and more than one-half million deaths each year. We evaluated the potential of tangerines and oranges to pre...

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

Targeting p53 by the small-molecule PRIMA-1(Met)/APR-246 has shown promising preclinical activity in various cancer types. However, the mechanism of PRIMA-1(Met)-induced apoptosis is not completely understood and its effect on multiple myeloma cells is unknown. In this study, we evaluated antitumor effect of PRIMA-1(Met) alone or its combination with current antimyeloma agents in multiple myeloma cell lines, patient samples, and a mouse xenograft model. Results of our study showed that PRIMA-1(Met) decreased the viability of multiple myeloma cells irrespective of p53 status, with limited cytotoxicity toward normal hematopoietic cells. Treatment of multiple myeloma cells with PRIMA-1(Met) resulted in induction of apoptosis, inhibition of colony formation, and migration. PRIMA-1(Met) restored wild-type conformation of mutant p53 and induced activation of p73 upregulating Noxa and downregulating Mcl-1 without significant modulation of p53 level. siRNA-mediated silencing of p53 showed a little effect on apoptotic response of PRIMA-1(Met), whereas knockdown of p73 led to substantial attenuation of apoptotic activity in multiple myeloma cells, indicating that PRIMA-1(Met)-induced apoptosis is, at least in part, p73-dependent. Importantly, PRIMA-1(Met) delayed tumor growth and prolonged survival of mice bearing multiple myeloma tumor. Furthermore, combined treatment of PRIMA-1(Met) with dexamethasone or doxorubicin displayed synergistic effects in both multiple myeloma cell lines and primary multiple myeloma samples. Consistent with our in vitro observations, cotreatment with PRIMA-1(Met) and dexamethasone resulted in enhanced antitumor activity in vivo. Our study for the first time shows antimyeloma activity of PRIMA-1(Met) and provides the rationale for its clinical evaluation in patients with multiple myeloma, including the high-risk group with p53 mutation/deletion.

Current and planned millimeter-wave passive and active satellite sensors are proposed for future low Earth-orbiting satellite platforms. For accurate modeling and sensor simulation of ice clouds at these wavelengths, realistic particle shapes and size distributions (PSD) need to be used. During the Jan-Feb 2012 Global Precipitation Measurement (GPM) Cold Season Precipitation Experiment (GCPEx) near Toronto, Canada, the Jet Propulsion Laboratory (JPL) dual-frequency (Ku/Ka-band) Airborne Precipitation Radar (APR-2) flew onboard the NASA DC-8 aircraft. Coordinated flights were carried out with the Univ. of North Dakota Citation aircraft carrying the High Volume Precipitation Spectrometer (HVPS-3), to collect cloud PSD and particle imagery. Selected flights enabled collection of coincident sampling volumes from the APR-2 and the HVPS. This unique dataset enables the scattering properties of the frozen hydrometeors to be modeled using the HVPS-provided particle distributions, and compared with APR-2 observations. The wide variety of fractal-like particle shapes measured in the HVPS data were separated into size bins and presented as 2 dimensional histograms with bins defined by Aspect (As) and Area (Ar) ratio. Individual histograms were previously characterized by the mean As and Ar values, ignoring the preferential linear trend between As and Ar visible for data points within most particle sizes. To facilitate the scattering models, we attempted to partition particles by shape within four size-invariant As and Ar categories. The four unique PSD, created by particle shape segregation, were then used to forward model the Ku and Ka- band radar reflectivities to locate the particle characteristics that provided the best agreement with actual APR-2 observations. In future work, these PSD will be used for passive microwave satellite sensor simulations of cold season precipitation and compared to actual satellite observations.

receiving end, demodulated data from the radio receiver is passed to the radio modem and is converted back to a digital data stream and decoded ...microcontroller parses the converts them into an APRS orwarded to the radio module nd a packet decoder on the a screen. Since it is only a us transmissions... decoded , the information is set to a layers component to be displayed. Below is an image representing various sources being plotted on World Wind

The termination of serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is regulated by its uptake by the 5-HT transporter (5-HTT), as well as its degradation by monoamine oxidase (MAO)-A. MAO-Adeficiency results in a wide set of behavioral alterations, including perseverative behaviors and social deficits. These anomalies are likely related to 5-HTergic homeostatic imbalances; however, the role of 5-HTT in these abnormalities remains unclear. To ascertain the role of 5-HTT in the behavioral anomalies associated to MAO-Adeficiency, we tested the behavioral effects of its blocker fluoxetine on perseverative, social and aggressive behaviors in transgenic animals with hypomorphic or null-allele MAO-A mutations. Acute treatment with 5-HTT blocker fluoxetine (10 mg/kg, i.p.) reduced aggressive behavior in MAO-A knockout (KO) mice and social deficits in hypomorphic MAO-ANeo mice. Furthermore, this treatment also reduced perseverative responses (including marble burying and water mist-induced grooming) in both MAO-A mutant genotypes. Both MAO-A mutant lines displayed significant reductions in 5-HTT expression across the prefrontal cortex, amygdala and striatum, as quantified by immunohistochemical detection; however, the down-regulation of 5-HTT in MAO-ANeo mice was more pervasive and widespread than in their KO counterparts, possibly indicating a greater ability of the hypomorphic line to enact compensatory mechanisms with respect to 5-HT homeostasis. Collectively, these findings suggest that the behavioral deficits associated with low MAO-A activity may reflect developmental alterations of 5-HTT within 5-HTergic neurons. Furthermore, the translational implications of our results highlight 5-HT reuptake inhibition as an interesting approach for the control of aggressive outbursts in MAO-Adeficient individuals. PMID:24882701

Concentrations of IgG2, IgG4 and IgE were low in 16, 24 and 20% of 25 persons with selective IgAdeficiency. Fifty-two per cent had IgD concentrations below 5 iu/ml. Trends for association between any of these parameters and the presence of clinical symptoms were not significant. All patients, except one, had normal amounts of Ig-bearing lymphocytes in the blood. IgG1 antibodies against casein were increased in titre and frequency, whereas IgG4 antibodies were normal. Similar results were found in other sera from persons with selective IgAdeficiency. PMID:3491694

The Alberta Pregnancy Outcomes and Nutrition (APrON) study is an ongoing prospective cohort study that recruits pregnant women early in pregnancy and, as of 2012, is following up their infants to 3 years of age. It has currently enrolled approximately 5000 Canadians (2000 pregnant women, their offspring and many of their partners). The primary aims of the APrON study were to determine the relationships between maternal nutrient intake and status, before, during and after gestation, and (1) maternal mood; (2) birth and obstetric outcomes; and (3) infant neurodevelopment. We have collected comprehensive maternal nutrition, anthropometric, biological and mental health data at multiple points in the pregnancy and the post-partum period, as well as obstetrical, birth, health and neurodevelopmental outcomes of these pregnancies. The study continues to follow the infants through to 36 months of age. The current report describes the study design and methods, and findings of some pilot work. The APrON study is a significant resource with opportunities for collaboration.

This paper presents the technical description, mechanical design, electronic components, software implementation and possible applications of a tele-operated mobile robot designed as an assisted living tool. This robotic concept has been named Assistant Personal Robot (or APR for short) and has been designed as a remotely telecontrolled robotic platform built to provide social and assistive services to elderly people and those with impaired mobility. The APR features a fast high-mobility motion system adapted for tele-operation in plain indoor areas, which incorporates a high-priority collision avoidance procedure. This paper presents the mechanical architecture, electrical fundaments and software implementation required in order to develop the main functionalities of an assistive robot. The APR uses a tablet in order to implement the basic peer-to-peer videoconference and tele-operation control combined with a tactile graphic user interface. The paper also presents the development of some applications proposed in the framework of an assisted living robot. PMID:27136552

A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed adeficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum. PMID:21599668

We purified a new extracellular serine proteinase (designated subtilisin Sendai) from the culture broth of alkalophilic Bacillus sp. G-825-6, and its properties were characterized. Its optimum pH was at 10.0, when succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-4-methylcoumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA) was used as a substrate. The substrate specificity of subtilisin Sendai was determined with oxidized insulin B-chain and fluorogenic peptidyl-MCA substrates. The isoelectric point of subtilisin Sendai was over 11.0. The molecular mass of the enzyme was estimated as 28,000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The circular dichroism spectrum of the enzyme was measured, and we discuss the relationship between the secondary structure of the enzyme and alkaline stability at pH 12 in comparison with that of subtilisin NAT. The structural gene (aprS) was cloned and sequenced. The deduced amino acid sequence for the mature protein (269 amino acids) was preceded by a putative signal sequence of 27 residues and a putative pro-sequence of 86 amino acids. The homology of the primary structure for 13 subtilisins was compared. The catalytic triad (Asp32, His64, and Ser221 with the numbering of subtilisin BPN') and the amino acid sequences near these amino acid residues were well conserved. As a special feature, it was observed that there was an extensive number of negatively charged amino acids in the pro-region of subtilisin Sendai and alkaline subtilisins. This was different from those of subtilisin from neutrophiles.

This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that adeficiency in cuticular wax synthesis and adeficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.

A case is reported of a 46 year old woman who died after a five month illness characterized by focal neurological signs, evidence of meningeal irritation, and the finding of `blast' cells in the CSF. Immunoglobulin A was consistently absent from her serum and secretions. Necropsy showed extensive infiltration by a reticulum-cell sarcoma of the subarachnoid space, with tumour nodules on several cranial nerves and tumour infiltration of the tuber cinereum. The significance of the association between immunoglobulin Adeficiency and neoplasia is discussed. Images PMID:4356731

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Plant-type APS reductase (APR), which catalyzes the reduction of activated sulfate to sulfite in plants, consists of a reductase domain and a C-terminal redox domain showing sequence homology to thioredoxin but possessing the activity of glutaredoxin. In order to understand the structural and biochemical properties of the redox domain of plant-type APS reductase, the C-terminal domain of APR1 (APR1C) from Arabidopsis thaliana was crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.70 Å on the SPXF beamline BL13B1 at the NSRRC, Taiwan. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 58.2, c = 86.7 Å. With one molecule per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.64 Å(3) Da(-1), which corresponds to a solvent content of approximately 53.49%. Further structure-based functional studies of APR1C would extend knowledge of the molecular mechanism and regulation of APR.

Après-coup finds its origins in Freud's earliest psychoanalytic writings, but it was only half a century ago that French psychoanalysts rediscovered, clarified, and developed the concept and so brought it recognition as an essential Freudian concept. Because the history of après-coup is embedded in the French reading of Freud, this article will give an account of that reading in relation to après-coup.

Immunohistochemistry for protein gene product 9.5 (PGP 9.5, a neuron specific protein) and vanilloid receptor 1-like receptor (VRL-1, a marker for medium-sized to large primary nociceptors) were used to assess the effects of Brn-3adeficiency on neuronal innervation of oral tissues and neurons of the trigeminal ganglion (TG). In the knockout mouse, the number of PGP 9.5-immunoreactive (-ir) nerve fibers decreased in the facial cutaneous and oral mucous epithelia, as well as the incisor and molar tooth germs. The reduction of PGP 9.5-ir Merkel endings was also observed in some vibrissae. No obvious change was detected in other tissues. Cell size analysis demonstrated that the proportion of small neurons markedly increased while that of medium-sized and large neurons significantly decreased in the TG of the mutant. Moreover, Brn-3adeficiency caused the disappearance of TG neurons which were immunoreactive for VRL-1. Together, our data suggest that nociceptors and low-threshold mechanoreceptors with medium-sized to large cell bodies may be sensitive to the loss of Brn-3a.

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Safety limits are required to maintain the integrity of physical barriers designed to prevent the uncontrolled release of radioactive materials in nuclear power plants. The safety analysis establishes two critical constraints that include an analytical limit in terms of a measured or calculated variable, and a specific time after the analytical limit is reached to begin protective action. Keeping with the nuclear regulations and industry standards, satisfying these two requirements will ensure that the safety limit will not be exceeded during the design basis event, either an anticipated operational occurrence or a postulated accident. Various studies on the setpoint determination methodology for the safety-related instrumentation have been actively performed to ensure that the requirement of the analytical limit is satisfied. In particular, the protection setpoint methodology for the advanced power reactor 1400 (APP1400) and the optimized power reactor 1000 (OPR1000) has been recently developed to cover both the design basis event and the beyond design basis event. The developed setpoint methodology has also been quantitatively validated using specific computer programs and setpoint calculations. However, the safety of nuclear power plants cannot be fully guaranteed by satisfying the requirement of the analytical limit. In spite of the response time verification requirements of nuclear regulations and industry standards, it is hard to find the studies on the systematically integrated methodology regarding the response time evaluation. In cases of APR1400 and OPR1000, the response time analysis for the plant protection system is partially included in the setpoint calculation and the response time test is separately performed via the specific plant procedure. The test technique has a drawback which is the difficulty to demonstrate completeness of timing test. The analysis technique has also a demerit of resulting in extreme times that not actually possible. Thus

The nucleotide sequences of two subgenomic RNA segments from influenza virus A/PR/8/34 have been determined by cloning viral cDNA into the vector M13mp7. Sequence analysis was facilitated by a re-cloning strategy which takes advantage of both wild-type and amber derivatives of the M13 vector. The RNA species (444 and 480 nucleotides) contain the 5' and 3' termini of segment 1 and therefore derive by simple internal deletions of this segment. However, these species are not exact copies of the terminal regions of the progenitor segment but contain a few base changes. These differences suggest that after these RNAs have arisen, their sequences can drift, presumably reflecting a lower selective pressure than on the standard RNA segments. PMID:7335495

The full-scope simulator is being developed to evaluate specific design feature and to support the iterative design and validation in the Man-Machine Interface System (MMIS) design of Advanced Power Reactor (APR) 1400. The simulator consists of process model, control logic model, and MMI for the APR1400 as well as the Power Control System (PCS). In this paper, a use case driven approach is proposed to develop a simulation model for PCS. In this approach, a system is considered from the point of view of its users. User's view of the system is based on interactions with the system and the resultant responses. In use case driven approach, we initially consider the system as a black box and look at its interactions with the users. From these interactions, use cases of the system are identified. Then the system is modeled using these use cases as functions. Lower levels expand the functionalities of each of these use cases. Hence, starting from the topmost level view of the system, we proceeded down to the lowest level (the internal view of the system). The model of the system thus developed is use case driven. This paper will introduce the functionality of the PCS simulation model, including a requirement analysis based on use case and the validation result of development of PCS model. The PCS simulation model using use case will be first used during the full-scope simulator development for nuclear power plant and will be supplied to Shin-Kori 3 and 4 plant. The use case based simulation model development can be useful for the design and implementation of simulation models. (authors)

Selective immunoglobulin Adeficiency (IgAD) is the most common primary immunodeficiency in Caucasians. It has previously been suggested to be associated with a variety of concomitant autoimmune diseases. In this review, we present data on the prevalence of IgAD in patients with Graves disease (GD), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), celiac disease (CD), myasthenia gravis (MG) and rheumatoid arthritis (RA) on the basis of both our own recent large-scale screening results and literature data. Genetic factors are important for the development of both IgAD and various autoimmune disorders, including GD, SLE, T1D, CD, MG and RA, and a strong association with the major histocompatibility complex (MHC) region has been reported. In addition, non-MHC genes, such as interferon-induced helicase 1 (IFIH1) and c-type lectin domain family 16, member A (CLEC16A), are also associated with the development of IgAD and some of the above diseases. This indicates a possible common genetic background. In this review, we present suggestive evidence for a shared genetic predisposition between these disorders. PMID:21826374

Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin Adeficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications. PMID:25389900

This short review summarizes all the published and unpublished reports on vitamin Adeficiency in the Sudan in the last four decades. Different local terms used by people to indicate vitamin Adeficiency were enlisted. There is evidence that vitamin Adeficiency is a public health problem in eastern Sudan and among communities from western and southern Sudan living around Greater Khartoum, who were displaced from their homelands because of drought, famine conditions and civil unrest. There are reports indicative of vitamin Adeficiency problem in the central and the far western provinces. There were no reports from the northern provinces. The need for a surveillance system was discussed.

This study focused on the relationship between students' Advanced Placement Program[R] (AP[R]) performance in AP English Language, Biology, Calculus, and U.S. History, and their subsequent college success. For each AP Exam studied, students were divided into three groups according to their AP Exam performance (no AP Exam taken, score of 1 or 2,…

This study was designed to determine if prenatal transportation influences the acute phase response (APR) to a postnatal Lipopolysaccharide (LPS) challenge. Pregnant Brahman cows (n=96) matched by age and parity were separated into transported (TRANS; n=48; transported for 2 hours on gestational day...

This study focused on the relationship between students' Advanced Placement Program[R] (AP[R]) performance in AP English Language, Biology, Calculus, and U.S. History, and their subsequent college success. For each AP Exam studied, students were divided into three groups according to their AP Exam performance (no AP Exam taken, score of 1 or 2,…

Curriculum surveys are used periodically to validate the use of Advanced Place Program[R] (AP[R]) Exams as assessments of skills required for advanced college-level study. In the case of AP German Language, the target level is third-year college language study. This article presents results from a 2003 curriculum survey completed by third-year…

This study documented the experiences of staging Vaslav Nijinsky's "L'Apres-midi d'un Faune" in a higher education dance program. The ballet was staged from Labanotation. Research questions focused on teaching and learning pedagogy, characterization of the process over time, experiences of the participants and teaching approaches. The project…

This study documented the experiences of staging Vaslav Nijinsky's "L'Apres-midi d'un Faune" in a higher education dance program. The ballet was staged from Labanotation. Research questions focused on teaching and learning pedagogy, characterization of the process over time, experiences of the participants and teaching approaches. The project…

A specialized Mu transducing phage containing a gene encoding ampicillin resistance and the lac structural genes without the lac promotor [Mu d(apr lac)] has been constructed and used to create gene fusions in Escherichia coli (M. J. Cadadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530--4533, 1979). Transposition of the Mu d(Apr lac) phage to chromosomal sites can result in lac expression being controlled by a chromosomal promoter. We have constructed an Escherichia coli K-12 strain in which the Mu d(Apr lac) phage is integrated into an F factor. The F+::Mu d(Apr lac) was then transferred by conjugation into a Salmonella typhimurium strain that was sensitive to L-arabinose. Strains containing gene fusions were selected as L-arabinose-resistant colonies after partial induction of the phage. Two classes of ara-lac fusion strains were isolated: (i) araC-lac fusions in which the expression of beta-galactosidase synthesis was constitutuve and not inducible by L-arabinose; and ((ii) fusion of the lac genes to the ara structural genes in which the expression of beta-galatosidase synthesis was induced 263-fold by L-arabinose.

The dissimilatory adenosine-5'-phosphosulfate (APS) reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers) catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP); in sulfur-oxidizing bacteria (SOB) it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures). Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. Based on the comparative models, there are no significant structural differences between

Background The dissimilatory adenosine-5′-phosphosulfate (APS) reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers) catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP); in sulfur-oxidizing bacteria (SOB) it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. Methodology/Principal Findings Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8–99.3% and 89.5–96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 Å with respect to the template structures). Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1) SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2) crenarchaeal SRP Caldivirga and Pyrobaculum, and (3) SOB of the distinct Apr lineage I) and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. Conclusions Based on the comparative models

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation. PMID:9755196

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10−8 M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens. PMID:16597964

We report the ocular and systemic manifestations of vitamin Adeficiency in a child with a complicated medical history including autism and a restricted diet, living in a developed country. This child had significant vitamin Adeficiency despite being under long-term medical care, yet the diagnosis was not considered until he had an ophthalmology review for visual deterioration.

Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that adeficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na(+), (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na(+)/K(+) homeostasis and hormonal balance.

Recent studies suggest that tocopherols could play physiological roles in salt tolerance but the mechanisms are still unknown. In this study, we analyzed changes in growth, mineral and oxidative status in vte1 and vte4 Arabidopsis thaliana mutants exposed to salt stress. vte1 and vte4 mutants lack α-tocopherol, but only the vte1 mutant is additionally deficient in γ-tocopherol. Results showed that adeficiency in vitamin E leads to reduced growth and increased oxidative stress in hydroponically-grown plants. This effect was observed at early stages, not only in rosettes but also in roots. The vte1 mutant was more sensitive to salt-induced oxidative stress than the wild type and the vte4 mutant. Salt sensitivity was associated with (i) high contents of Na+, (ii) reduced efficiency of PSII photochemistry (Fv/Fm ratio) and (iii) more pronounced oxidative stress as indicated by increased hydrogen peroxide and malondialdeyde levels. The vte 4 mutant, which accumulates γ- instead of α-tocopherol showed an intermediate sensitivity to salt stress between the wild type and the vte1 mutant. Contents of abscisic acid, jasmonic acid and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid were higher in the vte1 mutant than the vte4 mutant and wild type. It is concluded that vitamin E-deficient plants show an increased sensitivity to salt stress both in rosettes and roots, therefore indicating the positive role of tocopherols in stress tolerance, not only by minimizing oxidative stress, but also controlling Na+/K+ homeostasis and hormonal balance. PMID:23299430

We present new results of fully general relativistic magnetohydrodynamic (GRMHD) simulations of binary neutron star (BNS) mergers performed with the Whisky code. All the models use a piecewise polytropic approximation of the APR4 equation of state (EOS) for cold matter, together with a ''hybrid'' part to incorporate thermal effects during the evolution. We consider both equal and unequal-mass models, with total masses such that either a supramassive NS or a black hole (BH) is formed after merger. Each model is evolved with and without a magnetic field initially confined to the stellar interior. We present the different gravitational wave (GW) signals as well as a detailed description of the matter dynamics (magnetic field evolution, ejected mass, post-merger remnant properties, disk mass). Our new simulations provide a further important step in the understanding of these GW sources and their possible connection with the engine of short gamma-ray bursts (both in the ``standard'' and in the ``time-reversal'' scenarios) and with other electromagnetic counterparts.

We present new results of fully general relativistic magnetohydrodynamic simulations of binary neutron star (BNS) mergers performed with the Whisky code. All the models use a piecewise polytropic approximation of the APR4 equation of state for cold matter, together with a ‘hybrid’ part to incorporate thermal effects during the evolution. We consider both equal and unequal-mass models, with total masses such that either a supramassive NS or a black hole is formed after merger. Each model is evolved with and without a magnetic field initially confined to the stellar interior. We present the different gravitational wave (GW) signals as well as a detailed description of the matter dynamics (magnetic field evolution, ejected mass, post-merger remnant/disk properties). Our simulations provide new insights into BNS mergers, the associated GW emission and the possible connection with the engine of short gamma-ray bursts (both in the ‘standard’ and in the ‘time-reversal’ scenarios) and other electromagnetic counterparts.

A core catcher is adopted in the EU-APR1400 reactor design for management and mitigation of severe accidents with reactor core melting. The core catcher concept incorporates a number of engineering solutions used in the catcher designs of European EPR and Russian WER-1000 reactors, such as thin-layer corium spreading for better cooling, retention of the melt in a water-cooled steel vessel, and use of sacrificial material (SM) to control the melt properties. SM is one of the key elements of the catcher design and its performance is critical for melt retention efficiency. This SM consists of oxide components, but the core catcher also includes sacrificial steel which reacts with the metal melt of the molten corium to reduce its temperature. The paper describes the required properties of SM. The melt retention capability of the core catcher can be confirmed by modeling the heat fluxes to the catcher vessel to show that it will not fail. The fulfillment of this requirement is demonstrated on the example of LBLOCA severe accident. Thermal and physicochemical interactions between the oxide and metal melts, interactions of the melts with SM, sacrificial steel and vessel, core catcher external cooling by water and release of non-condensable gases are modeled. (authors)

The purpose of the study was to investigate whether acute strenuous exercise (1600- to 2500-m race) would elicit an acute phase response (APR) in Standardbred trotters. Blood levels of several inflammatory markers [serum amyloid A (SAA), haptoglobin, fibrinogen, white blood cell count (WBC), and iron], muscle enzymes [creatinine kinase (CK) and aspartate transaminase (AST)], and hemoglobin were assessed in 58 Standardbred trotters before and after racing. Hemoglobin levels increased and iron levels decreased 12 to 14 h after racing and haptoglobin concentrations, white blood cell counts, and iron levels were decreased 2 and/or 7 d after racing. Concentrations of CK, AST, SAA, and fibrinogen were unaltered in response to racing. Acute strenuous exercise did not elicit an acute phase reaction. The observed acute increase in hemoglobin levels and decreases in haptoglobin and iron levels may have been caused by exercise-induced hemolysis, which indicates that horses might experience a condition similar to athlete's anemia in humans. The pathogenesis and clinical implications of the hematological and blood-biochemical changes elicited by acute exercise in Standardbred trotters in the present study warrant further investigation.

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.

A spontaneous mutant of the cyanobacterium Synechocystis PCC6803 was isolated for its resistance to acetazolamide, an inhibitor of carbonic anhydrase. The mutant showed adeficiency in oxygen exchange between CO2 and H2O, a lower level of stable internal CO2 pool and a decreased capacity to adapt its photosynthetic affinity under limited inorganic carbon regime. The initial rate of uptake of inorganic carbon was identical to that of wild-type cells. It is demonstrated that the mutation affects the carbonic anhydrase activity. This could result from either of two impairments: adeficiency in the enzyme activity detectable by mass spectrometric determinations, or a modification of the cellular compartment in which the enzyme is located, preventing its activity. PMID:16667618

Betulinic acid, a pentacyclic triterpene isolated from Jujube tree (Zizyphus jujuba Mill), has been known for a wide range of biological and medicinal properties such as antibacterial, antimalarial, anti-inflammatory, antihelmintic, antinociceptive, and anticancer activities. In the study, we investigated the antiviral activity on influenza A/PR/8 virus infected A549 human lung adenocarcinoma epithelial cell line and C57BL/6 mice. Betulinic acid showed the anti-influenza viral activity at a concentration of 50 μM without a significant cytotoxicity in influenza A/PR/8 virus infected A549 cells. Also, betulinic acid significantly attenuated pulmonary pathology including increased necrosis, numbers of inflammatory cells and pulmonary edema induced by influenza A/PR/8 virus infection compared with vehicle- or oseltamivir-treated mice in vivo model. The down-regulation of IFN-γ level, which is critical for innate and adaptive immunity in viral infection, after treating of betulinic acid in mouse lung. Based on the obtained results, it is suggested that betulinic acid can be the potential therapeutic agent for virus infection via anti-inflammatory activity. PMID:26157551

The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp10----Cys (three cases), Tyr57----Asn, Asp85----Glu, Asp06----Asn (three cases), Asp96----Gly, Trp138----Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.

The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. PMID:26453908

Emphasis in adult education on qualifications and competence stigmatizes disadvantaged groups with adeficient identity. However, cultural studies indicate that people are competent developers of their own identities, who contribute to social life in a wide variety of contexts. (SK)

Carnitine palmitoyltransferase-1A (CPT-1A) deficiency is a defect of fatty acid metabolism that presents as an autosomal recessive inheritance. Carnitine palmitoyltransferase-1A is the rate-limiting enzyme that allows the body to process fats to provide energy during times of fasting and illness. Patients usually present between birth and 18 months of age following an illness with various symptoms including hypoketotic hypoglycemia, lethargy, and seizures. Diagnosis can be achieved through newborn metabolic screening. Long-term treatment is managed through dietary management. A milder form has been found to occur at a much higher incidence in the Inuit population. Since the recent discovery of CPT-1Adeficiency, much is yet to be learned. Researchers are busy identifying and studying groups of people who are presenting with CPT-1Adeficiency at significantly higher rates than the general population. This research will lead to a better understanding and future care of individuals diagnosed with CPT-1Adeficiency.

We report a 17-year-old boy who presented with a chronic left unilateral optic neuropathy. Computerized tomography and magnetic resonance imaging demonstrated compression of the left optic nerve due to skull hyperostosis. He was found to be profoundly vitamin Adeficient secondary to an unusual diet consisting predominantly of potato chips and crisps. Skull hyperostosis with cranial neuropathies and other neurological abnormalities has been described in growing animals fed vitamin Adeficient diets but has not been previously reported in humans.

Vitamin Adeficiency is among major health problems worldwide that leads to blindness, retarded growth and death, particularly in developing countries. In these countries, vitamin Adeficiency largely affects pre-school children, pregnant and lactating mothers, and the rural poor. For instance, the predicted prevalence of vitamin Adeficiency for 36 sub-Saharan African countries is 19.1%. Different strategies, including vitamin A supplementation, food fortification and dietary diversification, have been used to combat this problem. However, these strategies are not sustainable due to their high costs. Orange-fleshed sweet potato (Ipomoea batatas L. Lam) is a low priced crop, which is part of staple foods in most of sub-Saharan Africa that can be a year-round source of vitamin A. Most of the orange-fleshed sweet potato varieties contain 3000-16000 μg 100 g(-1) of β-carotene and this contributes to 250 to 1300 μg 100 g(-1) Retinol Activity Equivalents (RAE). Therefore, by using orange-fleshed sweet potato, it is possible to improve vitamin A status, increase the bio-availability of different micro-nutrients such as Fe, Zn, Ca and Mg, reduce vitamin Adeficiency and hence reduce child mortality rates by 23 to 30%. The article highlights the significance of vitamin A for human nutrition, the effect of vitamin Adeficiency, the different prevention methods and the potential of orange- fleshed sweet potato as a food crop to prevent vitamin Adeficiency.

The authors isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits adeficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. The authors have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

Plants contain an extensive family of PsbP-related proteins termed PsbP-like (PPL) and PsbP domain (PPD) proteins, which are localized to the thylakoid lumen. The founding member of this family, PsbP, is an established component of the Photosystem II (PS II) enzyme, and the PPL proteins have also been functionally linked to other photosynthetic processes. However, the functions of the remaining seven PPD proteins are unknown. To elucidate the function of the PPD5 protein (At5g11450) in Arabidopsis, we have characterized a mutant T-DNA insertion line (SALK_061118) as well as several RNAi lines designed to suppress the expression of this gene. The functions of the photosynthetic electron transfer reactions are largely unaltered in the ppd5 mutants, except for a modest though significant decrease in NADPH dehydrogenase (NDH) activity. Interestingly, these mutants show striking plant developmental and morphological defects. Relative to the wild-type Col-0 plants, the ppd5 mutants exhibit both increased lateral root branching and defects associated with axillary bud formation. These defects include the formation of additional rosettes originating from axils at the base of the plant as well as aerial rosettes formed at the axils of the first few nodes of the shoot. The root-branching phenotype is chemically complemented by treatment with the synthetic strigolactone, GR24. We propose that the developmental defects observed in the ppd5 mutants are related to adeficiency in strigolactone biosynthesis. PMID:22174848

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for adeficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

The antimicrobial power of honey seems to be ascribable to several factors, including oxidative and osmotic stress. The aim of this study was to find genetic determinants involved in the response to honey stress in the opportunistic pathogen Pseudomonas aeruginosa, chosen as model micro-organism. A library of transposon mutants of P. aeruginosa PAO1 was constructed and only four mutants unable to grow in presence of fir honeydew honey were selected. All four mutants were impaired in the major H2O2-scavenging enzyme catalase A (KatA). The knockout of katA gene caused sensitivity, as expected, not only to hydrogen peroxide but also to different types of honey including Manuka GMO 220 honey. Genetic complementation, as well as the addition of PAO1 supernatant containing extracellular catalase, restored tolerance to honey stress in all the mutants. As P. aeruginosa PAO1 catalase KatA copes with H2O2 stress, it is conceivable that the antimicrobial activity of honey is, at least partially, due to the presence of hydrogen peroxide in honey or the ability of honey to induce production of hydrogen peroxide. The katA-deficientmutants could be used as tester micro-organisms to compare the power of different types of natural and curative honeys in eliciting oxidative stress mediated by hydrogen peroxide.

We studied the immune response and the colonization pattern in vitamin A-deficient rats that were colonized with the Escherichia coli O6 K13 pOmp 21 strain, which is genetically manipulated to produce ovalbumin and to be resistant to ampicillin. In the vitamin A-deficient rats, the number of bacteria per gram of feces was about five times higher than in the paired fed control rats 4 weeks after colonization. In the control rats, the colon and the lower part of the ileum were colonized, while in the vitamin A-deficient rats all parts of the small intestine, as well as the colon, were heavily inhabited by bacteria. Furthermore, in 75% of the vitamin A-deficient rats, the E. coli bacteria were found in the mesenteric lymph nodes, and in 50% of the rats E. coli were found in the kidneys. These animals also developed severe arthritis. The levels of serum immunoglobulin G (IgG), IgM, IgE, and biliary IgA antibodies against the bacterial antigens were significantly higher in the vitamin A-deficient rats than in the control rats. The number of IgA-producing cells in the lamina propria of the small intestine was significantly lower in the vitamin A-deficient rats than in the control rats; however, there was an increase in the number of CD8+ cells and transforming growth factor beta-producing cells in the lamina propria of the vitamin A-deficient rats. Disturbances in T-cell function were demonstrated, since spleen cells from the vitamin A-deficient rats produced more gamma interferon and interleukin-2 in vitro than control spleen cells. In summary, vitamin Adeficiency led to a decrease in the ability to control the localization of intestinal bacteria and an increase in translocation, which was followed by development of arthritis regardless of substantial levels of antibacterial antibodies. The bacterial invasion made the animals hyperresponsive to the bacterial antigens, despite the fact that vitamin Adeficiency is normally associated with suppressed antibody production

Background Despite growing evidence that supports the importance of 6-month exclusive breastfeeding, few Canadian mothers adhere to this, and early weaning onto solids is a common practice. This study assessed infant feeding transitions during the first 6 months postpartum and factors that predicted exclusive breastfeeding to 3 and 6 months. Methods This prospective cohort study was part of the Alberta Pregnancy Outcomes and Nutrition study (APrON). From an initial sample of 600 pregnant women recruited from Edmonton and Calgary, 402 mothers provided complete details at 3 months postpartum; 300 stayed on to provide information at 6 months postpartum. During pregnancy and at 3 and 6 months postpartum, data on maternal and infant socio-demographic, behavior, and feeding were collected. Results Even though there was a high rate of “ever having breastfed” (98.6%), exclusive breastfeeding rates for 3 and 6 months were 54.0% and 15.3%, respectively. After controlling for potential confounders, the study showed that mothers who held post-graduate university degrees were 3.76 times more likely to breastfeed exclusively for 6 months than those without a university degree (95% CI: 1.30-10.92; p = 0.015). In addition, mother of previous children were more likely to breastfeed exclusively for 6 months (OR: 2.21, 95% CI: 1.08-4.52; p = 0.031). Mothers who were in the highest quartile of the Iowa Infant Feeding Attitude Score were 4.29 and 5.40 times more likely to breastfeed exclusively for 3 months (95% CI: 1.31-14.08; p-trend

In accord with the nomenclature proposed for mutants of simian virus 40 the same rules, with minor modifications, are recommended for naming mutants of adenoviruses. It is further suggested that these rules, which pertain to a system of classification based primarily upon complementation analysis, also be applied to mutants of other DNA-containing animal viruses. PMID:4355864

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

Following the successful Precipitation Radar (PR) of the Tropical Rainfall Measuring Mission, a new airborne, 14/35 GHz rain profiling radar, known as Airborne Precipitation Radar - 2 (APR-2), has been developed as a prototype for an advanced, dual-frequency spaceborne radar for a future spaceborne precipitation measurement mission. . This airborne instrument is capable of making simultaneous measurements of rainfall parameters, including co-pol and cross-pol rain reflectivities and vertical Doppler velocities, at 14 and 35 GHz. furthermore, it also features several advanced technologies for performance improvement, including real-time data processing, low-sidelobe dual-frequency pulse compression, and dual-frequency scanning antenna. Since August 2001, APR-2 has been deployed on the NASA P3 and DC8 aircrafts in four experiments including CAMEX-4 and the Wakasa Bay Experiment. Raw radar data are first processed to obtain reflectivity, LDR (linear depolarization ratio), and Doppler velocity measurements. The dataset is then processed iteratively to accurately estimate the true aircraft navigation parameters and to classify the surface return. These intermediate products are then used to refine reflectivity and LDR calibrations (by analyzing clear air ocean surface returns), and to correct Doppler measurements for the aircraft motion. Finally, the the melting layer of precipitation is detected and its boundaries and characteristics are identifIed at the APR-2 range resolution of 30m. The resulting 3D dataset will be used for validation of other airborne and spaceborne instruments, development of multiparametric rain/snow retrieval algorithms and melting layer characterization and statistics.

Following the successful Precipitation Radar (PR) of the Tropical Rainfall Measuring Mission, a new airborne, 14/35 GHz rain profiling radar, known as Airborne Precipitation Radar - 2 (APR-2), has been developed as a prototype for an advanced, dual-frequency spaceborne radar for a future spaceborne precipitation measurement mission. . This airborne instrument is capable of making simultaneous measurements of rainfall parameters, including co-pol and cross-pol rain reflectivities and vertical Doppler velocities, at 14 and 35 GHz. furthermore, it also features several advanced technologies for performance improvement, including real-time data processing, low-sidelobe dual-frequency pulse compression, and dual-frequency scanning antenna. Since August 2001, APR-2 has been deployed on the NASA P3 and DC8 aircrafts in four experiments including CAMEX-4 and the Wakasa Bay Experiment. Raw radar data are first processed to obtain reflectivity, LDR (linear depolarization ratio), and Doppler velocity measurements. The dataset is then processed iteratively to accurately estimate the true aircraft navigation parameters and to classify the surface return. These intermediate products are then used to refine reflectivity and LDR calibrations (by analyzing clear air ocean surface returns), and to correct Doppler measurements for the aircraft motion. Finally, the the melting layer of precipitation is detected and its boundaries and characteristics are identifIed at the APR-2 range resolution of 30m. The resulting 3D dataset will be used for validation of other airborne and spaceborne instruments, development of multiparametric rain/snow retrieval algorithms and melting layer characterization and statistics.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity. Images PMID:6874648

This case study reveals the analytical characterisation carried out on 'D'Aprés Cormon' (1891) by Veloso Salgado, a copy of 'Caïn' (1880) by Fernando Cormon, belonging nowadays to Musée d'Orsay. Infrared Reflectography made in situ revealed underdrawing grid that was used to transpose the original painting to a smaller scale, pictorial style, execution method and retouched/restored areas. In situ EDXRF analysis together with Raman microscopy allowed the identification of gypsum, lead white, titanium white, yellow ochre, vermilion, ultramarine and lamp black. These results provide valuable information about the Salgado's palette and his production technique.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP. PMID:25905574

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by adeficiency in activity for malate dehydrogenase. PMID:16347083

The non-lethal tomato mutant «lutescent-2» shows an early yellowing of normal developed leaves. Its ripe fruits display a yellow colouring, red pigment synthesis being delayed by up to two weeks. Typical pigment synthesis, related to leaf maturation, does not occur in mutant leaves. Both the concentration of chl a and chl b start to decrease very quickly at the end of leaf expansion. Early yellowing of «1-2» leaves appears to be related to the reduced car(470) content, which leads to chlorophyll photooxidation. Structural evidence of adeficiency in car(470) content in young «1- 2» plastids is given by a reduction of stroma thylakoids as well as by a limited grana stacking. The altered balance between the two pigment classes determined an active, even if incomplete, conversion of chloroplasts to chromoplast-like organelles.

The study was conducted to determine the demographic and health-related risk factors of subclinical vitamin Adeficiency in Ethiopia. Blood samples were collected from 996 children in 210 clusters across the nation for analysis of serum retinol. Interviews were conducted with the respective mothers of the 996 children on presumed risk factors of vitamin Adeficiency. A higher subclinical vitamin Adeficiency was associated with: not receiving vitamin A supplement over the year, having been ill during the two weeks preceding the survey, no or incomplete vaccination, belonging to a mother with high parity, and low levels of awareness of vitamin A. Moreover, being from Muslim household was strongly associated with higher levels of subclinical vitamin Adeficiency. Among the risk factors identified, low levels of vaccination, high parity, and low levels of maternal awareness of vitamin A contributed to higher risks of vitamin Adeficiency among Muslim children. The findings underscore the need for creation of strengthened awareness of family planning and importance of vitamin A, promotion of vaccination and child health, intensification of vitamin A supplementation, and in-depth investigation on factors contributing to increased vulnerability of Muslim children.

The most common humoral immunodeficiency is IgAdeficiency. One of the first papers addressing the cellular and molecular mechanisms underlying IgAdeficiency indicated that immature IgA-positive B-lymphocytes are present in these patients. This suggests that the genetic background for IgA is still intact and that class switching can take place. At this moment, it cannot be ruled out that genetic as well as environmental factors are involved. Areas covered: A clinical presentation, the biological functions of IgA, and the management of IgAdeficiency are reviewed. In some IgAdeficient patients, a relationship with a loss-of-function mutation in the TACI (transmembrane activator and calcium-modulating cyclophilin ligand interaction) gene has been found. Many other genes also have been associated. Gut microbiota are an important environmental trigger for IgA synthesis. Expert commentary: Expression of IgAdeficiency is due to both genetic and environmental factors and a role for gut microbiota cannot be excluded.

Hereditary muscular dystrophic chickens of the Storrs strain possess two genetic disorders, muscular dystrophy (MD) and adeficient concanavalin A (Con A), a T-cell mitogen, mediated splenic lymphocyte blastogenic response. A possible amelioration of the MD phenotype in MD chickens expressing normal Con A was postulated on the basis of progeny segregating for these two traits in F2 genetic analyses. To test this possibility, testcross progeny were examined for segregation of MD and Con Adeficiency traits, and for the degree of muscle destruction and Con Adeficiency. The data show both traits to be inherited independently as autosomal recessive traits, and do not support any phenotypic modifications occurring in chickens expressing MD with normal Con A. In the testcross progeny, the Con Adeficiency disorder is equally deficient in normal and MD progeny, and the degree of muscle destruction as measured by serum creatine phosphokinase levels is equally great in MD chickens with or without the Con Adeficiency trait. The reduced numbers of MD chickens in the testcross progeny can be accounted for by chance and probably reflect losses during in ovo development.

Previous work from the laboratory has shown by immunochemical assay that vitamin Adeficiency caused a greatly increased plasma level of fibronectin (FN), a high molecular-weight glycoprotein involved in cell adhesion and found in plasma. The authors determined that primary hepatocytes of vitamin A-deficient rats secreted more FN into the medium than those of pair-fed controls, synthesized more FN (as measured by uptake of /sup 35/S-methionine) and had a higher level of mRNA specific for FN. If, however, they measured uptake of /sup 3/H-glucosamine into FN secreted by hepatocytes from vitamin A-deficient rats, they found a decrease in glycosylation of FN, as determined by the /sup 35/S-methionine to /sup 3/H-glucosamine ratio in the secreted FN. Since underglycosylated FN has a higher turnover than the glycosylated form, the authors hypothesized that in vitamin A-deficient hepatocytes, the increased breakdown of underglycosylated FN caused increased synthesis of the protein portion of the molecule by a feedback mechanism. To test this hypothesis, they incubated hepatocytes from normal rats with tunicamycin. They found that /sup 3/H-glucosamine uptake was almost completely suppressed, but there was a relative increased uptake of /sup 35/S-methionine. This result suggested that in vitamin A-deficient liver cells, decreased glycosylation may be the cause of the increased synthesis of the protein portion of FN.

Homeobox-containing genes may play an important role in establishing embryonic patterns during development of vertebrates. Retinoic acid is able to induce expression of Hox genes in cells in culture and to alter expression patterns in the developing vertebrate embryos. Using wholemount in situ hybridization, we have examined and compared the expression patterns of a homeobox-containing gene, Msx-1, in early normal and vitamin A-deficient quail embryos. At gastrulation stage, Msx-1 is primarily expressed in the posterior half of both normal and vitamin A-deficient embryos. However, the gene is expressed wider and stronger in the vitamin A-deficient embryos. At neurulation stages, Msx-1 is continuously expressed in the posterior region up to Hensen's node and in the edge of the neural fold in both normal and vitamin A-deficient embryos. Notably, in the vitamin A-deficient embryos, Msx-1 is expressed more strongly and is also expressed ectopically in the anterior and precardiac regions. These results provide evidence that endogenous retinoids are involved in the normal expression of Msx-1 in avian embryo and that the expression of Msx-1 is downregulated by endogenous and physiological retinoids in vivo during early avian embryogenesis.

High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption. PMID:25559441

High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region. The disruption of an APR that lies at the interface of light and heavy chain variable domains, VH and VL, via L45K mutation destabilized MAB 1 and abolished antigen binding. However, mutation at the preceding residue (V44K), which also lies in the same APR, increased apparent solubility and reduced viscosity of MAB 1 without sacrificing antigen binding or thermal stability. Neutralizing the negatively charged surface patch (E59Y) also increased apparent solubility and reduced viscosity of MAB 1, but charge reversal at the same position (E59K/R) caused destabilization, decreased solubility and led to difficulties in sample manipulation that precluded their viscosity measurements at high concentrations. Both V44K and E59Y mutations showed similar increase in apparent solubility. However, the viscosity profile of E59Y was considerably better than that of the V44K, providing evidence that inter-molecular interactions in MAB 1 are electrostatically driven. In conclusion, neutralizing negatively charged surface patches may be more beneficial toward reducing viscosity of highly concentrated antibody solutions than charge reversal or aggregation prone motif disruption.

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products. PMID:28217561

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin Adeficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin Adeficiency and should be an excellent animal model of vitamin Adeficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

The three mutant alleles of the ABA locus of Arabidopsis thaliana result in plants that are deficient in the plant growth regulator abscisic acid (ABA). The authors have used {sup 18}O{sub 2} to label ABA in water-stressed leaves of mutant and wild-type Arabidopsis. Analysis by selected ion monitoring and tandem mass spectrometry of ({sup 18}O)ABA and its catabolites, phaseic acid and ABA-glucose ester ({beta}-D-glucopyranosyl abscisate), indicates that the aba genotypes are impaired in ABA biosynthesis and have a small ABA precursor pool of compounds that contain oxygens on the rings, presumably oxygenated carotenoids (xanthophylls). Quantitation of the carotenoids form mutant and wild-type leaves establishes that the aba alleles cause adeficiency of the epoxy-carotenoids violaxanthin and neoxanthin and an accumulation of their biosynthetic precursor, zeaxanthin. These results provide evidence that ABA is synthesized by oxidative cleavage of epoxy-carotenoids (the indirect pathway). Furthermore the carotenoid mutant they describe undergoes normal greening. Thus the aba alleles provide an opportunity to study the physiological roles of epoxy-carotenoids in photosynthesis in a higher plants.

Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. Adeficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant, L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site. Km and Vmax values for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apo-carboxyl carrier protein as a substrate. A Km for biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. The Vmax for the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.

The cells of an aggregateless mutant of Dictyostelium discoïdeum, agip 235, can cooperate with other aggregateless or wild strains to form differentiated aggregates. A soluble mediator liberated by the coaggregating cells seems responsible for the development of agip 235. In most cases, the development of mutant agip 235 stops at the aggregation stage; however, its coaggregation with the mutant 518 results in cosporulation, with the production of viable spores of each genotype, effecting a phenotypic suppression of both mutations.

Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1-6 photoreceptors, and a mutant of the ninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodopsin content in R1-6 photoreceptors is reduced by 10(2)-10(6)-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologically normal. Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model. Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps. Mutant bumps are strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletion of rhodopsin content, rather than to any specific alterations in the opsin protein. Lowering or buffering the intracellular calcium concentration by EGTA injection mimics the effects of the mutations on the bump amplitude, but, unlike the mutations, it also affects the latency and kinetics of light responses. PMID:3097245

Vitamin Adeficiency (VAD) is an important cause of blindness. Red palm oil (RPO) is the richest food source of VA-forming carotenoids. We evaluated RPO carotenoid concentration and bioavailability, and used this data to estimate the amount of RPO needed to meet VA requirements. Amounts ranged fr...

A study of 40 individuals (ages 5-31) with autism investigated immune deficiency. Results found the subjects had significantly lower serum immunoglobulin A (IgA) levels than that of age- and sex-matched controls. Seventeen subjects had one of three haplotypes associated with IgAdeficiency and seven of these had decreased IgA levels. (CR)

A study of 40 individuals (ages 5-31) with autism investigated immune deficiency. Results found the subjects had significantly lower serum immunoglobulin A (IgA) levels than that of age- and sex-matched controls. Seventeen subjects had one of three haplotypes associated with IgAdeficiency and seven of these had decreased IgA levels. (CR)

Although humoral immune mechanisms are currently thought to be of pathogenetic significance in juvenile rheumatoid arthritis (JRA), little is known about the role of cellular hypersensitivity in this disease. A possible association between abnormalities of humoral and cellular immunity exists in patients with ataxia-telangiectasia, who may have absent IgA, abnormal delayed hypersensitivity, or both. As IgAdeficiency has been noted in 2–3% of patients with JRA, we have studied selected aspects of humoral and cellular hypersensitivity in patients with JRA and IgAdeficiency and in patients with JRA and normal IgA levels. All patients had normal serum levels of complement, IgG, IgM, and IgD. Cellular hypersensitivity was evaluated by cutaneous delayed-type hypersensitivity, in vitro migration inhibitory factor production, and antigen induced 3H-thymidine incorporation by lymphocytes using Candida and Streptokinase–Streptodornase antigens. Two of four IgAdeficient patients had positive in vitro but negative in vivo responses to antigens. Seven of fourteen JRA patients with normal immunoglobulin levels exhibited a similar dissociation of in vivo and in vitro manifestations of delayed hypersensitivity. This pattern of cellular immune response was associated with activity and chronicity of disease; it was independent of IgAdeficiency. PMID:5022450

Immunoglobulin Adeficiency is the most common primary immunodeficiency defined as decreased serum level of IgA (less than 7 mg/dl) in the presence of normal levels of other immunoglobulin isotypes. Most individuals with IgAdeficiency are asymptomatic and identified coincidentally. However, some patients may present with recurrent infections, allergic disorders and autoimmune manifestations, such as diabetes mellitus, Graves disease and celiac disease. The international literature has not produced any kind of review yet about intra-oral manifestations of selective IgA-deficiency. L.S., a 7-year-old Caucasian girl, was examined at our hospital. After she had undergone a professional dental cleaning, a symmetric, bilateral ulcerative gingivitis developed nearby the upper second primary molars. The gingival ulcers were persistent and did not disappear in the following 3 weeks. In the meantime, the young patient reported the presence of gastrointestinal symptoms. IgA serum level was 4.5 mg/dl, while the other isotypes levels were in the common range. The diagnosis of selective IgA-deficiency was formulated and the girl underwent further examination for the specific IgG autoantibodies in celiac disease, which were not present. Consequently, a full prevention program was planned. This case report emphasizes the role of the paediatric dentist in the early detection of systemic disorder, such as the immunological diseases. The oral cavity often reveals to be the first site of manifestation of important systemic diseases. Immunoglobulin A (IgA) deficiency is the most common primary immunodeficiency and is defined as a decrease in serum IgA levels in the presence of normal levels of other immunoglobulin isotypes (1). Serum IgAdeficiency was first described in children with ataxia-telangiectasia (2) and has since been identified in other patients, including normal patients. The prevalence of IgAdeficiency ranges from 1:223 to 1:1000 in community studies and from 1:400 to 1

Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. PMID:26951227

Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment

A rare mutant allele (cad-n1) of the cad gene in loblolly pine (Pinus taeda L.) causes adeficiency in the production of cinnamyl alcohol dehydroganase (CAD). Effects associated with this allele were examined by comparing wood density and growth traits of cad-n1 heterozygous trees with those of wild-type trees in a 10-year-old open-pollinated family...

Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.'' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies. PMID:8056325

For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell. PMID:10511552

FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

Several mutants have been used in pairwise crosses to determine complementation Accessionl For VTIS G!RA& DTIC T %B 3 fl!, -r,. AD) Av ’Di [ Annual...Ghendon (1973) indicate that a large number of polio virus ts mutants producing a pathologic change in infected monkeys were assayed for virus produccion

Five Arabidopsis mutants have been isolated on the basis of hypersensitivity of leaf tissue to UV light. For each mutant, the UV-hypersensitive phenotype (uvh) was inherited as a single recessive Mendelian trait. In addition, each uvh mutant represented a separate complementation group. Three of the mutations producing the UV hypersensitive phenotype have been mapped relative to either genetic markers or physical microsatellite polymorphisms. Locus UVH1 is linked to nga76 on chromosome 5, UVH3 to GL1 on chromosome three, and UVH6 to nga59 on chromosome 1. Each uvh mutant has a characteristic pattern of sensitivity based on UV sensitivity of leaf tissue, UV sensitivity of root tissue, and ionizing radiation sensitivity of seeds. On the basis of these patterns, possible molecular defects in these mutants are discussed. 30 refs., 3 figs., 5 tabs.

Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

To explore the risk of infections in individuals with IgAdeficiency compared to general population controls. In this nationwide prospective population-based cohort study, we used data on IgA levels (<0.07 g/L) from six university hospitals in Sweden to identify 2100 individuals with IgAdeficiency. Individuals were diagnosed between 1980 and 2010. For each patient with IgAdeficiency we identified 10 controls from the general population, matched on age, sex, and place of residence (n = 18,653). Data on infections were obtained from the Swedish National Patient Register (including inpatient and hospital-based outpatient care) between 2001 and 2010. We defined infections as having a record of a relevant international classification of disease (ICD) code. Prevalences and prevalence ratios (PRs) were calculated. Individuals with IgAdeficiency were more likely to have a record of any infection (36.1 vs. 18.8% in controls) corresponding to a PR of 2.4 (95%CI 2.2-2.6). We also noted statistically significant associations with IgAdeficiency (all P-values <0.05) and respiratory tract infections (17.8 vs. 6.3% in controls; PR = 3.2), gastrointestinal infections (6.0 vs. 1.8% in controls; PR = 3.5), skin infections (4.1 vs. 2.2% in controls; PR = 1.9), joint infections (0.48 vs. 0.24% in controls; PR = 2.0; P = 0.052), sepsis (1.5 vs. 0.45% in controls; PR = 3.4), meningitis (0.38 vs. 0.12%, PR = 3.2), mastoiditis/otitis (2.1 vs. 1.1% in controls; PR = 2.0), and urinary tract infections (6.1 vs. 3.4% in controls; PR = 1.8). Individuals with IgAdeficiency are at an increased risk of infections requiring hospital care.

... Power Reserve (APR), an Automatic Takeoff Thrust Control System (ATTCS), for Go-Around Performance... airplane will have novel or unusual design features associated with utilizing go-around performance credit...: Federal eRegulations Portal: Go to http://www.regulations.gov/ and follow the online instructions for...

This document is a synthesis of the key points provided in the Office of Special Education Programs (OSEP) Early Childhood Transition FAQs: SPP/APR indicators C-8 and B-12 released on December 1, 2009. It was developed to assist states with the implementation of effective transition policies and practices. The following is included: (1) Transition…

The study objective was to determine if surgical castration at birth or weaning impacts the acute phase response (APR) or complete blood counts (CBC) and whether concurrent administration of an oral analgesic (meloxicam) ameliorates inflammation. Bull calves (n=29) from the University of Arkansas re...

... 15 Commerce and Foreign Trade 2 2014-01-01 2014-01-01 false U.S. Government requests for information needed to satisfy the requirements of the APR or the Act. 781.4 Section 781.4 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY...

Vitamin Adeficiency has rarely been reported in captive or free-ranging wildlife species. Necropsy findings in two captively housed southern sea otters (Enhydra lutris nereis) included irregular thickening of the calvaria characterized by diffuse hyperostoses on the internal surface. One animal also had moderate squamous metaplasia of the seromucinous glands of the nose. There was no measurable retinol in the liver of either sea otter. For comparison, hepatic retinol concentration was determined for 23 deceased free-ranging southern and northern (Enhydra lutris kenyoni) sea otters from California and Alaska. Free-ranging otters were found to have similar hepatic retinol concentrations (316 +/- 245 mg/kg wet weight) regardless of their location and subspecies. All of these values were significantly higher than the levels in the affected animals. Consumption of a diet with very low vitamin A concentrations and noncompliance in daily supplementation are hypothesized as the causes of vitamin Adeficiency in these two sea otters.

We report a 42-year-old woman, who presented with proteinuria (3.85 g/day) and malleolar oedema. She had a medical history of Graves' disease, recurrent upper respiratory tract infections, episodes of Raynaud phenomenon and dysphagia. Biochemistry showed a selective IgAdeficiency (SIgAD). Percutaneous renal needle biopsy showed diffuse global thickening of the glomerular basement membranes on light microscopy and granular deposits of IgG and C3 along the glomerular basement membranes on immunofluorescence. The pathological diagnosis was membranous glomerulopathy stage II. A treatment with dietary salt restriction and an angiotensin-converting enzyme inhibitor was initiated, resulting in a reduction of proteinuria. Despite the fact that selective IgAdeficiency is associated with various autoimmune disorders, the association with glomerular disease is rather rare and the pathogenesis is not fully understood.

A 32 year old woman presented with acute onset of abdominal pain and fever. An urgent computerised tomography (CT) of the whole abdomen showed dilated loop at the terminal ileum in the right lower abdomen with thickening of the wall and oedema. The CT was suggestive of distal small bowel obstruction at the ileum with surrounding wall oedema. Multiple biopsies taken from the terminal ileum and colon on colonoscopy were all unremarkable. She represented one-year later with a recurrence of intestinal obstruction. CT enteroclysis showed collapse at the distal 3 cm segment of the terminal ileum. There was no associated wall thickening, active inflammatory changes or ileitis. This was suspicious of post-inflammatory change or fibrosis. She was subsequently found to have selective IgAdeficiency with recurrent infection in the terminal ileum resulting in intestinal obstruction. In conclusion, selective IgAdeficiency should be considered in patients with recurrent intestinal obstruction without anatomical obstructions. PMID:28090187

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

Nearly half of women of reproductive age (WRA) in Cambodia are anemic. To guide interventions, national data on nutritional causes of anemia, including iron deficiency and vitamin Adeficiency, are needed. In 2012, a national household survey in WRA on antibodies to routine vaccine-preventable disease immunity was performed. We used serum samples from this survey to estimate the prevalence of iron and vitamin Adeficiency in 2112 Cambodian WRA, aged 15 to 39 years. Iron deficiency was classified as low or marginal iron stores (ferritin concentrations corrected for inflammation <15 μg/L and <50 μg/L respectively; Fer), iron deficient erythropoiesis (soluble transferrin receptor concentrations >8.3 mg/L; sTfR), or low total body iron (TBI) derived from Fer and sTfR concentrations (<0 mg/kg). Vitamin A status was classified using retinol binding protein (RBP) concentrations corrected for inflammation as deficient (<0.70 μmol/L) or marginal (<1.05 μmol/L. Overall, the prevalence of low iron stores, low TBI and iron deficient erythropoiesis was 8.1%, 5.0% and 9.3% respectively. Almost 40% of the women had marginal iron stores. Iron status was better in women living in urban areas compared to rural areas (p < 0.05 for TBI and sTfR). The prevalence of vitamin Adeficiency was <1%. These findings suggest that the contribution of iron and vitamin Adeficiency to the high prevalence of anemia in Cambodian WRA may be limited. The etiology of anemia in Cambodia needs to be elucidated further to guide current policies on anemia. PMID:27043624

Nearly half of women of reproductive age (WRA) in Cambodia are anemic. To guide interventions, national data on nutritional causes of anemia, including iron deficiency and vitamin Adeficiency, are needed. In 2012, a national household survey in WRA on antibodies to routine vaccine-preventable disease immunity was performed. We used serum samples from this survey to estimate the prevalence of iron and vitamin Adeficiency in 2112 Cambodian WRA, aged 15 to 39 years. Iron deficiency was classified as low or marginal iron stores (ferritin concentrations corrected for inflammation <15 μg/L and <50 μg/L respectively; Fer), iron deficient erythropoiesis (soluble transferrin receptor concentrations >8.3 mg/L; sTfR), or low total body iron (TBI) derived from Fer and sTfR concentrations (<0 mg/kg). Vitamin A status was classified using retinol binding protein (RBP) concentrations corrected for inflammation as deficient (<0.70 μmol/L) or marginal (<1.05 μmol/L. Overall, the prevalence of low iron stores, low TBI and iron deficient erythropoiesis was 8.1%, 5.0% and 9.3% respectively. Almost 40% of the women had marginal iron stores. Iron status was better in women living in urban areas compared to rural areas (p < 0.05 for TBI and sTfR). The prevalence of vitamin Adeficiency was <1%. These findings suggest that the contribution of iron and vitamin Adeficiency to the high prevalence of anemia in Cambodian WRA may be limited. The etiology of anemia in Cambodia needs to be elucidated further to guide current policies on anemia.

Maintenance of a surface immune barrier is important for homeostasis in organs with mucosal surfaces that interface with the external environment; however, the role of the mucosal immune system in chronic lung diseases is incompletely understood. We examined the relationship between secretory IgA (SIgA) on the mucosal surface of small airways and parameters of inflammation and airway wall remodeling in chronic obstructive pulmonary disease (COPD). We studied 1,104 small airways (<2 mm in diameter) from 50 former smokers with COPD and 39 control subjects. Small airways were identified on serial tissue sections and examined for epithelial morphology, SIgA, bacterial DNA, nuclear factor-κB activation, neutrophil and macrophage infiltration, and airway wall thickness. Morphometric evaluation of small airways revealed increased mean airway wall thickness and inflammatory cell counts in lungs from patients with COPD compared with control subjects, whereas SIgA level on the mucosal surface was decreased. However, when small airways were classified as SIgA intact or SIgAdeficient, we found that pathologic changes were localized almost exclusively to SIgA-deficient airways, regardless of study group. SIgA-deficient airways were characterized by (1) abnormal epithelial morphology, (2) invasion of bacteria across the apical epithelial barrier, (3) nuclear factor-κB activation, (4) accumulation of macrophages and neutrophils, and (5) fibrotic remodeling of the airway wall. Our findings support the concept that localized, acquired SIgAdeficiency in individual small airways of patients with COPD allows colonizing bacteria to cross the epithelial barrier and drive persistent inflammation and airway wall remodeling, even after smoking cessation.

Vitamin Adeficiency (VAD) after bariatric surgery is recognised as a significant post-operative complication that can lead to visual impairment. We report two cases of night blindness and visual impairment caused by VAD after malabsorptive bariatric surgery. Both patients were treated with intramuscular vitamin A replacement and made near complete recovery in their vision. Ocular complications due to VAD should be diagnosed and treated promptly in patients after bariatric surgery because these complications are reversible.

The present cross sectional study was carried out to estimate the prevalence of vitamin Adeficiency among children by means of clinics and conjunctival impression cytology (CIC), and nutritional status by anthropometric indicators H//A, W//A, W//H. The study population included 157 children 2-6 y old, from urban and rural slums of Maracaibo, Venezuela, Conjunctival impression cytology was performed by ICEPO standard procedure. Z-score was applied to anthropometric data with reference values of NCHS-WHO. No evidence of clinical or ophthalmologic signs of vitamin Adeficiency were detected. The prevalence of subclinical vitamin Adeficiency, as detected by abnormal CIC, was 35.4%, being higher in rural children (48.3%). These prevalence values are higher than the criteria laid down by WHO/UNICEF to indicate a public health problem (> 20%). Mild or moderate protein-energy global malnutrition and stunting were detected in 36.1% and 44.6% of children, respectively. Abnormal CIC was indistinctly observed (approximately equal to 35%) as much in children with adequate nutrition as in malnourished ones. There was no significant difference in the distribution of the CIC results in relation to nutritional status. The findings indicate that CIC and Z-score of nutritional anthropometric data are useful to characterize the risk of vitamin Adeficiency and of malnutrition in communities. Beside the implementation of an integral nutritional program which includes supplementation, food fortification and dietary diversification, improvement of socio-economic and sanitation conditions and also the educational level, with emphasis on nutritional and health education, are highly recommended.

To report a successfully treated case of acquired night blindness associated with fundus white spots secondary to vitamin Adeficiency. An ocular examination, electrophysiologic testing, as well as visual field and OCT examinations were obtained on a 61-year-old man with vitamin Adeficiency who had previously undergone gastric bypass surgery. The patient had a re-evaluation after treatment with high doses of oral vitamin A. The patient was observed to have numerous white spots in the retina of each eye. Best-corrected visual acuity was initially 20/80 in each eye, which improved to 20/40-1 OU after oral vitamin A therapy for 2 months. Full field electroretinogram (ERG) testing, showed non-detectable rod function and a 34 and 41% reduction for 32-Hz flicker and single flash cone responses, respectively, below the lower limits of normal. Both rod and cone functions markedly improved after initiation of vitamin A therapy. Vitamin Adeficiency needs to be considered in a patient with white spots of the retina in the presence of poor night vision.

OBJECTIVE: To determine the prevalence of vitamin Adeficiency in children aged 6-9 years in northern Ethiopia. METHODS: A cross-sectional study was carried out and the data were analysed for 824 (61.5%) of 1339 eligible children for whom there was complete information on biochemical vitamin A status, dietary vitamin A intake, ocular examination for xerophthalmia, and anthropometry. FINDINGS: The prevalence of xerophthalmia was 5.8%; serum retinol levels were below 0.35 mumol/l and between 0.35 and 0.70 mumol/l in 8.4% and 51.1% of the children respectively. The liver vitamin A reserve (modified relative dose response ratio > or = 0.06) was low in 41.0% of the children. CONCLUSION: The high prevalence of severe vitamin Adeficiency in children aged 6-9 years indicates the need to reevaluate the practice of targeting vitamin A supplementation programmes on children under 6 years of age in areas where vitamin Adeficiency is endemic. PMID:11417037

Biliopancreatic diversion (BPD) is a malabsorptivebariatric procedure can lead to the development of several nutritional complications, including fat-soluble vitamins deficiencies. Routine supplementation with vitamins and trace elements and a close follow-up long-term can prevent these nutritional risks. Vitamin A participates in ocular metabolism, epithelial differentiation, growth, and embryogenesis. Have been described several cases of ophthalmological and fetal complications associated with vitamin Adeficiency in patients who have undergone BPD. Few information exists in literature about dermatologic manifestations that may occur in these patients. Phrynoderma is a type of follicular hyperkeratosis located on the extensor surfaces of the extremities whose main cause is vitamin Adeficiency. We report an exceptional case of severe cutaneous and ocular complications in a patient who had undergone BPD with poor adherence to treatment and postoperative follow-up. Our patient presented simultaneously the characteristic skin lesions of phrynoderma with nytalopia and xerophthalmia in a setting of low serum levels of vitamin A. Treatment with high doses vitamin A obtained the resolution of both processes. We review and discuss the relationship between phrynoderma, malnutrition and vitamin Adeficiency.

Introduction. Few data are available on vitamin Adeficiency in the gastric bypass population. Methods. We performed a retrospective chart review of gastric bypass patients (n = 69, 74% female). The relationship between serum vitamin A concentration and markers of protein metabolism at 6-weeks and 1-year post-operative were assessed. Results. The average weight loss at 6-weeks and 1-year following surgery was 20.1 ± 9.1 kg and 44.1 ± 17.1 kg, respectively. At 6 weeks and 1 year after surgery, 35% and 18% of patients were vitamin Adeficient, (<325 mcg/L). Similarly, 34% and 19% had low pre-albumin levels (<18 mg/dL), at these time intervals. Vitamin A directly correlated with pre-albumin levels at 6 weeks (r = 0.67, P < 0.001) and 1-year (r = 0.67, P < 0.0001). There was no correlation between the roux limb length measurement and pre-albumin or vitamin A serum concentrations at these post-operative follow-ups. Vitamin A levels and markers of liver function testing were also unrelated. Conclusion. Vitamin Adeficiency is common after bariatric surgery and is associated with a low serum concentration of pre-albumin. This fat-soluble vitamin should be measured in patients who have undergone gastric bypass surgery and deficiency should be suspected in those with evidence of protein-calorie malnutrition.

Purpose To present a case of unusual corneal infection early in the course of peripheral ulcerative keratitis in a patient with severe vitamin Adeficiency. Method Single observational case report in urban USA. Case Presentation An alcoholic patient with pancreatitis, chronic diarrhea, and vitamin Adeficiency presented with a marginal corneal ulcer from which two bacteria of the family Micrococcaceae were cultured and identified by genome sequence analysis, namely Kocuria palustris and Rothia mucilaginosa. Soon after, severe bilateral peripheral ulcerative keratitis developed, later accompanied by eyelid cellulitis of one lid. These conditions improved with antibiotics, treatment of the underlying gastrointestinal conditions, and treatment of the vitamin deficiency. Conclusion Susceptibility to keratitis with unusual bacteria of the Micrococcaceae family can occur in the setting of alcoholism-related gastrointestinal disease with severe vitamin Adeficiency. To our knowledge, K. palustris is a species not previously identified in any human disease, and the Kocuria genus has not previously been reported as a participant in eye infection. Documented cases of R. mucilaginosa in ocular disease are rare. These unusual infections heralded the onset of severe marginal corneal melts. PMID:24707276

Immunoglobulin A (IgA) deficiency is the most common primary deficiency. We aimed to define the prevalence of IgAdeficiency among healthy school children in Turkey and the differences between geographical regions. Blood samples were collected from 20,331 healthy school children from all regions across Turkey. The serum IgA levels were tested through nephelometric method, and all 108 samples with IgA levels lower than 5 g/L were tested through ELISA for IgG and IgM levels. All IgG and IgM values were within the normal range in all cases, and no concomitant deficiency was observed. Our study results showed that the selective IgAdeficiency incidence was 0.52% (1:188). The highest incidence, of 1:128.7, was observed in children from the Marmara region; the Black Sea Region levels (1:132.7) were lower, and the Mediterranean levels (1:365.7) were the lowest.

NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

A Bacillus subtilis mutation (gene symbol fdpA1), producing adeficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes. Images PMID:6257649

The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene

Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures. PMID:6988386

Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

Mechanical and contractile properties of tibialis anterior (TA) muscles from X-linked muscular dystrophic (mdx) mutant mice at different stages of development are compared to those of muscles from normal control animals. There is no difference between the tension output, speeds of contraction and relaxation, and weight of TA muscles from mutant adults and normal control animals. However, it is found that in 3-4-week-old mutant animals, tension output and muscle weight are very much reduced, and half relaxation time is prolonged. Thus, during this stage of development, muscles from mdx mice do not function properly. Histological examination of these muscles provides further evidence that, in these animals, rapid muscle destruction occurs at a particular time of development and that it is followed by complete recovery. This new mutant therefore presents an interesting case of muscle destruction and rapid regeneration. However, it is not an adequate model for Duchenne muscular dystrophy.

Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...mutations found in breast cancer using both structural and cell based assays. We have now have evidence for the effects of the most recurrent

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

The quantitative analysis of genetic interactions between pairs of gene mutations has proven effective for characterizing cellular functions but can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed Triple Mutant Analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, that is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principle actors are deleted. TMA has also uncovered double mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete, and measures interactions for up to 30 double mutants against a library of 1536 single mutants. PMID:25010907

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation. Images PMID:2974801

A marked increase in concomitant autoimmune diseases has previously been noted in patients with myasthenia gravis (MG). We show that these diseases occur both before and after the onset of MG and that the process is not influenced by thymectomy. IgAdeficiency (IgAD), which is strongly associated with the same HLA haplotype as early onset MG, has recently been suggested to be an autoimmune disease. However, there was no increase in the prevalence of IgAD in a large cohort of Swedish MG patients. PMID:21669464

Background The prevalence of infections by nontuberculous mycobacteria (NTM) has steadily increased over the past decades, especially in immunocompromised patients. Case presentation We present a patient with IgA-deficiency and mixed cutaneous infection by two slowly growing mycobacteria, Mycobacterium (M.) haemophilum and M. kansasii. Conclusions Cutaneous M. haemophilum infections most often result from HIV or transplantation-associated immunosuppression. Rarely, M. haemophilum may also infect healthy patients or iatrogenically immunosuppressed patients without transplantation. M. kansasii is one of the most frequent NTM and large awareness exists about its involvement in human diseases. Mycobacterial diagnosis of cutaneous infections should be considered in long-lasting skin lesions. PMID:21269422

Recurrent parotitis is a non-obstructive, non-suppurative inflammatory disease which is characterized by unilateral or bilateral parotid gland swelling attacks. It is also known as juvenile recurrent parotitis. Although the etiology is unknown, congenital malformations of the ductus, genetic predisposition, infections, allergies, autoimmune diseases, and some immune deficiencies are blamed. Here, we present a case report of recurrent parotitis with selective immunoglobulin Adeficiency in a six-year-old boy. The patient was presented to us with a new episode of swelling of left parotid region. In the last 2 years, the patient suffered from recurrent parotitis which lasted for approximately 5 days in ten individual episodes. PMID:24883201

Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1-seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

Opioid use in HIV infection has been associated with an increased frequency of neurological disease and cognitive impairment and vitamin Adeficiency has been linked to progressive HIV disease in drug users. In this report the potential effects of these factors, alone and in combination, on gamma amino butyric acid (GABA)-expression interneurons in hippocampus in the HIV-1 transgenic rat (TG) model were studied. TG and wild-type (WT) F344 Fisher rats deficient in vitamin A from birth were implanted either with a 37.5 mg morphine tablet or with a matching placebo and total numbers of neurons and of parvalbumin+ neurons were quantitated and parvalbumin expression was quantitated in the CA1 hippocampal region of the rats. These studies showed that total neuronal numbers were decreased in the TG versus WT Fisher rats and that this decrease was enhanced by the vitamin Adeficient diet and by treatment with morphine. In contrast, there was no significant change noted in numbers of parvalbumin+ neurons. However, levels of parvalbumin expression were decreased for vitamin Adeficient and morphine-treated WT rats as compared to WT rats on the normal diet and placebo-treated WT rats. For TG rats, parvalbumin expression was higher for vitamin Adeficient TG rats treated with either placebo or morphine than for WT vitamin Adeficient rats treated with placebo, and placebo treated vitamin Adeficient TG rats showed higher expression than morphine treated vitamin Adeficient rats. Expression was also higher for vitamin Adeficient morphine-treated rats than for the corresponding WT rat groups and for vitamin Adeficient TG rats treated with placebo. For the remaining groups, parvalbumin was similar for the TG and WT rats. These findings suggest that in hippocampus vitamin Adeficiency and morphine can increase parvalbumin expression, perhaps as a manifestation of a stress response. Parvalbumin-expressing GABA-ergic interneurons regulate the primary neuronal output from

H-4 rat hepatoma cells were mutagenized with ethyl methane-sulfonate and the frequency of emergence of cAMP resistant mutant cells were evaluated by cloning the EMS-treated cells in a semi-solid agar medium that contained either 1-3 mM 8-bromo-cAMP plus 1 mM 3-isobutyl-1-methyl xanthine or 5 ..mu..g/ml cholera toxin plus 1 mM IBMX. cAMP resistant mutants emerged at a frequency of 8 x 10/sup -5/. 15 colonies were isolated, recloned, grown in mass culture, and cell extracts were prepared. Analysis of cAMP-dependent protein kinase demonstrated that: (1) the type II enzyme is the only cAMP-dependent protein kinase detected in extracts of the hepatoma cells; (2) of the 15 cAMP resistant clonal cell lines examined, only one (H/sub 4/M/sub 18/) was found to be devoid of cAMP-dependent protein kinase activity. In another cell line (H/sub 4/M/sub 10/) the activity was 30% of that of the parental H-4 cells; (3) there was an increase (130-300%) in cAMP-dependent protein kinase activity in 13/15 of the mutant cell lines over that of the parental H-4 cells. Analysis of cAMP-phosphodiesterase demonstrated significant increases (150-370%) in the enzyme activity in extracts of the mutants over that of the H-4 parental line. Their results suggest that while adeficiency in cAMP-dependent protein kinase may confer resistance to the hepatoma cells against the cytostatic effects of 8-bromo-cAMP and cholera toxin, other events such as overexpression of phosphodiesterase may contribute to this phenotype.

In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

A replica plating method for isolating it amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10(-3) per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.

Influenza viruses cause significant morbidity and mortality in humans through epidemics or pandemics. Currently, two classes of anti-influenza virus drugs, M2 ion-channel inhibitors (amantadin and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir), have been used for the treatment of the influenza virus infection. Since the resistance to these drugs has been reported, the development of a new antiviral agent is necessary. In this study, we examined the antiviral efficacy of the plant extracts against the influenza A/PR/8/34 infection. In vitro, the antiviral activities of the plant extracts were investigated using the cell-based screening. Three plant extracts, Thuja orientalis, Aster spathulifolius, and Pinus thunbergii, were shown to induce a high cell viability rate after the infection with the influenza A/PR/8/34 virus. The antiviral activity of the plant extracts also increased as a function of the concentration of the extracts and these extracts significantly reduced the visible cytopathic effect caused by virus infections. Furthermore, the treatment with T. orientalis was shown to have a stronger inhibitory effect than that with A. spathulifolius or P. thunbergii. These results may suggest that T. orientalis has anti-influenza A/PR/8/34 activity.

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed adeficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed adeficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

Selective IgAdeficiency (SIgAD) is the most prevalent immunodeficiency worldwide, progressing to common variable immunodeficiency only in few reported cases. We report the case of a Spanish female aged 22 and diagnosed of selective IgAdeficiency, a long history of bronchitis, several episodes of pneumonia, bilateral bronchiectasis, normal IgG, IgM, IgG subclasses, and detectable pre-vaccination IgG antibodies against tetanus toxoid and Streptococcus pneumoniae. She was evaluated in our clinic in order to rule out common variable immunodeficiency. We observed good antibody response to tetanus toxoid, absence of circulating switched memory B cells, decreased response to pneumococcal polysaccharide antigens and a lack of response to Salmonella typhi vaccine. Most SIgAD patients presents with upper respiratory tract infections or mild diarrhea. Those with lower tract infections, pneumonia or untreatable diarrhea should follow B-cell subpopulations' study and antibody response to vaccines. Absence of response to Salmonella typhi vaccine allowed us to expose the defective antibody production.

Visual symptoms (night blindness) of vitamin Adeficiency (VAD) were among the earliest diet-related deficiencies documented. Knowledge of vitamin A chemistry, metabolism and deficiency consequences accrued rapidly during the first eight decades of the 20th century. A series of disorders were described in animals, including impaired growth, reproduction, epithelial integrity, and disease resistance that were relieved by consumption of both animal and plant sources of the vitamin. Identification of the intestinal beta-carotene cleavage enzyme in the laboratory of James Allen Olson was seminal to understanding the mechanism for formation of vitamin A from ingested carotenoids. WHO's 1990 estimate of about 40 million children annually with clinical eye signs of VAD was revised upward to 140-250 million at risk of vitamin Adeficiency disorders (VADD) when epidemiological and clinical trials demonstrated morbidity and mortality risk even in the absence of ocular signs. Alternative methods for VAD status assessment and more reliable analytical techniques were developed, several in Dr. Olson's laboratory. The last decade has seen global progress in VADD control by expanding distribution of medicinal supplements, fortification of foods and dietary diversification through horticulture and education programs. Experience shows that achievements gained through narrowly focused interventions are fragile and vulnerable to national political and economic instability. Contextually relevant, community-centered strategies that improve household food and nutrition security and self-reliance are critical to sustaining international efforts to control the VADD "pox."

Endophilin-A, a well-characterized endocytic adaptor essential for synaptic vesicle recycling, has recently been linked to neurodegeneration. We report here that endophilin-Adeficiency results in impaired movement, age-dependent ataxia, and neurodegeneration in mice. Transcriptional analysis of endophilin-A mutant mice, complemented by proteomics, highlighted ataxia- and protein-homeostasis-related genes and revealed upregulation of the E3-ubiquitin ligase FBXO32/atrogin-1 and its transcription factor FOXO3A. FBXO32 overexpression triggers apoptosis in cultured cells and neurons but, remarkably, coexpression of endophilin-A rescues it. FBXO32 interacts with all three endophilin-A proteins. Similarly to endophilin-A, FBXO32 tubulates membranes and localizes on clathrin-coated structures. Additionally, FBXO32 and endophilin-A are necessary for autophagosome formation, and both colocalize transiently with autophagosomes. Our results point to a role for endophilin-A proteins in autophagy and protein degradation, processes that are impaired in their absence, potentially contributing to neurodegeneration and ataxia.

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-Adeficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg. Images PMID:1326642

A spontaneous dwarf mutant of groundnut variety, Kopergaon-3, showed differential expression for plant height and secondary branching characters in the reciprocal F1 populations. These differences were assumed to be due to the interaction of nuclear and cytoplasmic factors which mutated with dwarfness.Segregation for dwarfness in the F2 and F3 generations confirmed the monogenic inheritance. The mutant expression was, therefore, controlled by a pair of recessive factors designated d(v)d(v), indicating dwarfism in the Valencia group.

Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.

Our previous study demonstrated that Melaleuca alternifolia (tea tree) oil (TTO) had an interesting antiviral activity against Influenza A in MDCK cells. In fact, when we tested TTO and some of its components, we found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and alfa-terpineol were the main active components. The aim of this study was to investigate the mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1 in MDCK cells. None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Thus, the effect of TTO and its active components on different steps of the replicative cycle of influenza virus was studied by adding the test compounds at various times after infection. These experiments revealed that viral replication was significantly inhibited if TTO was added within 2h of infection, indicating an interference with an early step of the viral replicative cycle of influenza virus. The influence of the compound on the virus adsorption step, studied by the infective center assay, indicated that TTO did not interfere with cellular attachment of the virus. TTO did not inhibit influenza virus neuraminidase activity, as shown by the experiment measuring the amount of 4-methylumbelliferone, cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid. The effect of TTO on acidification of cellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control. The treatment of cells with 0.01% (v/v) of TTO at 37°C for 4h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles, indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

To investigate the prevalence of vitamin Adeficiency among lactating women in a poor urban population of Bangladesh, and to examine the relationship between various factors and vitamin A status. Cross-sectional study. Maternal and child health clinic in Dhaka City, Bangladesh. A total of 120 lactating women aged 17-37 years were randomly selected from women who attended a local maternal and child health clinic in Dhaka City for immunisation of their children. Various socio-economic, personal characteristics, dietary intakes of vitamin A and anthropometric data were collected. Serum retinol (vitamin A) concentration was determined as a measure of vitamin A status. Of the subjects, 37% had low serum vitamin A levels (<30 microg x dl-1), with 13.3% having sub-clinical vitamin Adeficiency (<20 microg x dl-1). Eighty-seven per cent had vitamin A intakes below the recommended dietary allowance. The lactating women who were either illiterate or received only informal education had significantly lower serum vitamin A levels compared with those who received formal education. The women whose husbands received formal education had significantly higher serum vitamin A levels than those whose husbands were either illiterate or received only informal education. The serum vitamin A levels of women in households with poor sanitation/latrine practice were significantly lower than those of women in households with good sanitation/latrine practice. The women with one child had significantly lower serum vitamin A levels than those with two or more children. Women with a lactation period of 6 months or more had significantly lower serum vitamin A levels than women with a lactation period of less than 6 months. The women who consumed less than the median vitamin A intake (274.8 microg day-1) had significantly lower serum vitamin A levels than those who consumed more than the median vitamin A intake. By multiple regression analysis, education level of the women, number of living

Vitamin A-deficient (A-) mice make strikingly poor IgG responses when they are immunized with purified protein antigens. Previously, we showed that A- T cells overproduce interferon gamma (IFN-gamma), which then could inhibit interleukin 4 (IL-4)-stimulated B cell IgG responses. To determine if the altered IFN-gamma regulation pattern and its immunological consequences would extend to a natural infection, we studied mice infected with the parasitic helminth Trichinella spiralis. The course of the infection was similar in A- and A-sufficient (A+) mice. These mice did not differ with respect to newborn larvae/female/hour produced in the intestine, or muscle larvae burden 5 wk postinfection. They also did not differ in the intestinal worm expulsion rate until day 15, when A- mice still harbored parasites, whereas A+ mice had cleared intestinal worms. Vitamin Adeficiency reduced both the frequency of B lymphocytes secreting IgG1 antibodies to parasite antigens, and the bone marrow eosinophilia associated with helminth infection. The cytokine secretion patterns in infected mice were consistent with these observations and with previous studies. Mesenteric lymph node cells from infected A- mice secreted significantly more IFN-gamma, and significantly less IL-2, IL-4, and IL- 5 than infected A+ controls. A- splenocytes secreted significantly more IFN-gamma, and equivalent amounts of IL-2, IL-4, and IL-5 compared with A+ controls. Interestingly, CD4-CD8- cells secreted the majority of the IL-4 produced in the spleen. The IL-2, IL-4, and IL-5 steady-state transcript levels correlated with secreted protein levels, but IFN- gamma transcripts did not. Although they secreted more protein, A- cells contained fewer IFN-gamma transcripts than A+ cells. These results suggest two vitamin A-mediated regulation steps in IFN-gamma gene expression: positive regulation of IFN-gamma transcript levels, and negative regulation posttranscriptionally. The essentially unaltered outcome of T

Background The exclusion of individuals with elevated acute phase proteins has been advocated in order to improve prevalence estimates of vitamin Adeficiency in surveys, but it is unclear whether this will lead to sampling bias. The purpose of the study was to determine whether the exclusion of individuals with elevated acute phase proteins is associated with sampling bias and to characterize inflammation in children with night blindness. Methods In a survey in the Republic of the Marshall Islands involving 281 children, aged 1–5 years, serum retinol, C-reactive protein (CRP), and α1-acid glycoprotein (AGP) were measured. Results Of 281 children, 24 (8.5%) had night blindness and 165 (58.7%) had serum retinol <0.70 μmol/L. Of 248 children with AGP and CRP measurements, 123 (49.6%) had elevated acute phase proteins (CRP >5 mg/L and/or AGP >1000 mg/L). Among children with and without night blindness, the proportion with serum retinol <0.70 μmol/L was 79.2% and 56.8% (P = 0.03) and with anemia was 58.3% and 35.7% (P = 0.029), respectively. The proportion of children with serum retinol <0.70 μmol/L was 52.0% after excluding children with elevated acute phase proteins. Among children with and without elevated acute phase proteins, mean age was 2.8 vs 3.2 years (P = 0.016), the proportion of boys was 43.1% vs. 54.3% (P = 0.075), with no hospitalizations in the last year was 11.0% vs 23.6% (P = 0.024), and with anemia was 43.8% vs 31.7% (P = 0.05), respectively. Conclusions Exclusion of children with inflammation in this survey of vitamin Adeficiency does not improve prevalence estimates for vitamin Adeficiency and instead leads to sampling bias for variables such as age, gender, anemia, and hospitalization history. PMID:15588289

Glossy mutants are a common occurrence in Brassica oleracea L. and they have been documented in most crop varieties of the species including cabbage, kale, broccoli, and collard. Glossy phenotypes have been of particular interest to researchers due to observations that they influence insect behavior...

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. PMID:14617778

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

A current theory of anxiety effects in cognition claims that anxiety disrupts normal processing within the working memory system. We examined this theory in the context of a reading task, for participants who were high or low in assessed mathematics anxiety. The task was designed to measure the ability to inhibit attention to distracting information and the effects of this ability on explicit memory performance. The results suggested that math-anxious individuals have adeficient inhibition mechanism whereby working memory resources are consumed by task-irrelevant distracters. A consequence of this deficiency was that explicit memory performance was poorer for high-anxious individuals. Based on these results, the recommendation is made that Eysenck and Calvo's (1992) processing efficiency theory be integrated with Connelly, Hasher, and Zack's (1991) inhibition theory to portray more comprehensively the relation between anxiety and performance.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked genetic disorder caused by adeficiency of iduronate 2-sulfatase (IDS), and missense mutations comprising about 30% of the mutations responsible for MPS II result in heterogeneous phenotypes ranging from the severe to the attenuated form. To elucidate the basis of MPS II from the structural viewpoint, we built structural models of the wild type and mutant IDS proteins resulting from 131 missense mutations (phenotypes: 67 severe and 64 attenuated), and analyzed the influence of each amino acid substitution on the IDS structure by calculating the accessible surface area, the number of atoms affected and the root-mean-square distance. The results revealed that the amino acid substitutions causing MPS II were widely spread over the enzyme molecule and that the structural changes of the enzyme protein were generally larger in the severe group than in the attenuated one. Coloring of the atoms influenced by different amino acid substitutions at the same residue showed that the structural changes influenced the disease progression. Based on these data, we constructed a database of IDS mutations as to the structures of mutant IDS proteins. PMID:27695081

As one of a number of p53-regulated genes, Gadd45a (growth arrest and DNA damage inducible gene) has been shown to delay carcinogenesis and decrease mutation frequency. Gadd45a is known to regulate nucleotide excision DNA repair (NER) in response to UV radiation. Here, we report an emerging role for Gadd45a in base excision repair (BER). Gadd45a-null mouse embryo fibroblasts MEF and gadd45a-deficient human colon cancer cells exhibited slow BER after treatment with methyl methanesulfonate (MMS) a pure base-damaging agent. In addition, removal of AP sites by apurinic/apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref1) was significantly delayed in gadd45a-null cells. Moreover, the localization of APE1/Ref1 within the nucleus was observed in gadd45a wild-type cells, whereas APE1 become mainly distributed in the cytoplasm, and there is a reduced interaction with proliferating cell nuclear antigen (PCNA) in Gadd45a-deficient cells. Inasmuch as p53 has been shown to regulate BER in addition to the NER pathway, our data suggest that p53-regulated gene Gadd45a contributes to the BER response by affecting the interaction of cellular APE1/Ref1 with PCNA. Gadd45a might be a key component gene of the p53 pathway involved in protection from carcinogenic base damage and maintenance of genomic stability, although the downstream mechanism including APE1/Ref1 will need further study.

A representative sample of 1510 preschool children living in the Bandiagra circle (Mopti Region, Mali) was examined between March and April 1997 to determine the level of vitamin Adeficiency. Using a randomized two level cluster sampling, 20 clusters of 75 children aged six months to six years were selected for evaluating xerophthalmia (XN night blindness and/or X1B Bitot spot). Concurrently stature and weight were determined. A semiquantitative seven-day dietary questionnaire was applied to the mothers of 484 infants to assess consumption of vitamin A rich foodstuffs. The prevalence of biochemical deficiency was attested using the Modified Relative Dose Response test (MRDR) on a sub-sample of 192. Of the studied children, 4.3% (95% Confidence interval [CI]: 3.2-5.3) reported night blindness and 2% (95% CI: 1.3-2.7) had Bitot spots. Prevalence of xerophthalmia attested by at least one of these signs was 5.4% (95% CI: 4.2-6.5). The prevalence reached 10.5% at three years of age. The MRDR test proved abnormal in 77.1% of the subjects (95% CI: 70.3-82.7). Serum retinol was lower than 0.35 micro mol/L in 43.8% (95.6% CI: 36.9-51.3) and less than 0.70 micro mol/L in 92.7% of the children (95% CI: 87.8-95.8). Weekly consumption of vitamin A rich food was rare: 75.8% had not eaten any animal vitamin A rich food, and 22.1% had consumed less than seven times a vitamin A rich food of either vegetable or animal origin. These data define vitamin Adeficiency as a severe public health problem in the Bandiagara area of Mali.

To clarify the relationship between hexosaminidase A (HEX A) activity and GM2-ganglioside hydrolysis in atypical clinical situations of HEX Adeficiency, we have developed a simple method to assess GM2-ganglioside metabolism in cultured fibroblasts utilizing GM2 labeled with tritium in the sphingosine portion of the molecule. The radioactive lipid is added to the media of cultured skin fibroblasts, and after 10 days the cells are thoroughly washed, then harvested, and their lipid composition analyzed by HPLC. The degree of hydrolysis of the ingested GM2 is determined by comparing the amount of radioactive counts recovered in undegraded substrate with total cellular radioactivity. Adeficiency in GM2-ganglioside hydrolysis was demonstrated in seven HEX A-deficient adults with neurological signs and in two healthy-appearing adolescents with older affected siblings. In each case, an analysis of endogenous monosialoganglioside composition revealed an increase in GM2-ganglioside, confirming the presence of a block in the metabolism of GM2. No defect in GM2-catabolism was found in four other healthy individuals with HEX Adeficiency. This method of assay is especially helpful in the evaluation of atypical cases of HEX Adeficiency for the definitive diagnosis of GM2-gangliosidosis.

To clarify the relationship between hexosaminidase A (HEX A) activity and GM2-ganglioside hydrolysis in atypical clinical situations of HEX Adeficiency, we have developed a simple method to assess GM2-ganglioside metabolism in cultured fibroblasts utilizing GM2 labeled with tritium in the sphingosine portion of the molecule. The radioactive lipid is added to the media of cultured skin fibroblasts, and after 10 days the cells are thoroughly washed, then harvested, and their lipid composition analyzed by HPLC. The degree of hydrolysis of the ingested GM2 is determined by comparing the amount of radioactive counts recovered in undegraded substrate with total cellular radioactivity. Adeficiency in GM2-ganglioside hydrolysis was demonstrated in seven HEX A-deficient adults with neurological signs and in two healthy-appearing adolescents with older affected siblings. In each case, an analysis of endogenous monosialoganglioside composition revealed an increase in GM2-ganglioside, confirming the presence of a block in the metabolism of GM2. No defect in GM2-catabolism was found in four other healthy individuals with HEX Adeficiency. This method of assay is especially helpful in the evaluation of atypical cases of HEX Adeficiency for the definitive diagnosis of GM2-gangliosidosis. Images Fig. 1 PMID:2934978

Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from 14C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in α-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation. Images PMID:4967197

Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

Vitamin A is an important constituent of the epidermis, where it plays a crucial role in epidermal turnover. Adeficiency of epidermal vitamin A may be the consequence of nutritional vitamin Adeficiency, exposure to sunlight or any UV source, oxidative stress or chronological ageing. As a consequence, any treatment aiming at increasing epidermal vitamin A would exert a protective effect against these deleterious conditions. Retinoids may counteract some deleterious actions of UV radiation by physical and biological mechanisms. Topical natural retinoic acid precursors such as retinaldehyde or retinol are less irritant than acidic retinoids and may prevent epidermal vitamin Adeficiency due to nutritional deficiency, exposure to sunlight or any condition leading to free radical production. Retinoids may be combined with other compounds with complementary actions against ageing, nutritional deficiency and cancer, such as antioxidants, to potentiate their beneficial effects in the skin.

Altogether 29 745 English blood donors were screened for IgAdeficiency by double diffusion analysis; 57 had apparent absence of IgA, a frequency of 1:522. Further examination by the more sensitive haemagglutination inhibition assay revealed 34 samples having no detectable IgA, a frequency of 1:875. All donors negative by double diffusion analysis were tested for the presence of antibodies to IgA. Six class specific anti IgA antibodies and four anti IgA antibodies of limited specificity were detected. Three of these had the specificity anti alpha2 and one anti A2m(2). The 34 IgAdeficient donors detected provide a source of IgAdeficient blood for transfusion to patients with anti IgA antibodies. PMID:304071

The hepatic CYP4A-dependent ω-hydroxylation of arachidonic acid and CYP2C11-dependent 2α-/16α-hydroxylations of testosterone were decreased to 74 and 60% of respective control in microsomal fractions from vitamin A-deficient rats. Decreases in the rates of arachidonic acid ω-1-hydroxylation and testosterone 6β-, 7α- and 17α-hydroxylations were less pronounced.Corresponding decreases in microsomal CYP4A and CYP2C11 immunoreactive protein expression to 64 and 68% of respective control were observed in vitamin A-deficient rat liver. Expression of CYP3A proteins was unchanged from vitamin A-adequate control.Northern analysis revealed a selective decrease in CYP4A2 mRNA expression in vitamin A-deficient rat liver to ∼5% of control; expression of the related CYP4A1/4A3 mRNAs was not decreased. CYP2C11 mRNA expression was also decreased in vitamin A-deficient male rat liver to 39% of control levels.Intake of the deficient diet containing all-trans-retinoic acid (ATRA) during the final week of the experiment restored CYP4A2 mRNA and CYP4A protein. Administration of exogenous androgen or episodic growth hormone was ineffective. In contrast, CYP2C11 expression was restored by ATRA and androgen, but not by growth hormone.From these studies it emerges that CYP4A2, a fatty acid ω-hydroxylase in rat liver, is highly dependent on vitamin A for optimal expression, whereas CYP2C11 is indirectly down regulated by androgen deficiency resulting from vitamin A-deficiency. Altered CYP expression in vitamin A-deficiency provides insights into the relationship between dietary constituents and the intracellular formation of vasoactive eicosanoids as well as the clearance of androgenic steroids. PMID:11724755

Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. Images PMID:1551829

Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures. PMID:907329

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

The European Mouse Mutant Archive (EMMA) offers the worldwide scientific community a free archiving service for its mutant mouse lines and access to a wide range of disease models and other research tools. EMMA is currently comprised of seven partners who operate as the primary mouse repository in Europe. EMMA' s primary objectives are to establish and manage a unified repository for maintaining mouse mutations and to make them available to the scientific community. In addition to these core services, the consortium can generate germ-free (axenic) mice for its customers and also hosts courses in cryopreservation. EMMA is a founder member of the Federation of International Mouse Resources (FIMRe). The EMMA network is funded by the participating institutes, national research programmes and the European Commission Research Infrastructures Programme.

The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs

The dissimilatory adenosine-5′-phosposulfate reductase is a key enzyme of the microbial sulfate reduction and sulfur oxidation processes. Because the alpha- and beta-subunit-encoding genes, aprBA, are highly conserved among sulfate-reducing and sulfur-oxidizing prokaryotes, they are most suitable for molecular profiling of the microbial community structure of the sulfur cycle in environment. In this study, a new aprA gene-targeting assay using a combination of PCR and denaturing gradient gel electrophoresis is presented. The screening of sulfate-reducing and sulfur-oxidizing reference strains as well as the analyses of environmental DNA from diverse habitats (e.g., microbial mats, invertebrate tissue, marine and estuarine sediments, and filtered hydrothermal water) by the new primer pair revealed an improved microbial diversity coverage and less-pronounced template-to-PCR product bias in direct comparison to those of the previously published primer set (B. Deplancke, K. R. Hristova, H. A. Oakley, V. J. McCracken, R. Aminov, R. I. Mackie, and H. R. Gaskins, Appl. Environ. Microbiol. 66:2166-2174, 2000). The concomitant molecular detection of sulfate-reducing and sulfur-oxidizing prokaryotes was confirmed. The new assay was applied in comparison with the 16S rRNA gene-based analysis to investigate the microbial diversity of the sulfur cycle in sediment, seawater, and manganese crust samples from four study sites in the area of the Lesser Antilles volcanic arc, Caribbean Sea (Caribflux project). The aprA gene-based approach revealed putative sulfur-oxidizing Alphaproteobacteria of chemolithoheterotrophic lifestyle to have been abundant in the nonhydrothermal sediment and water column. In contrast, the sulfur-based microbial community that inhabited the surface of the volcanic manganese crust was more complex, consisting predominantly of putative chemolithoautotrophic sulfur oxidizers of the Betaproteobacteria and Gammaproteobacteria. PMID:17921272

The influence of vitamin Adeficiency on the development of cellular immune responses was examined using vitamin A-deficient mice (A mice)/Hymenolepis nana system. Mesenteric lymph node cells (MLNC) prepared from both normal BALB/c mice and A mice during H. nana infection proliferated extensively when cultured with soluble egg antigen of H. nana as assessed by 3H-thymidine incorporation. MLNC from normal mice secreted significantly more IL-2 and significantly less IFN-gamma than A mice when the cells were cultured in the presence of soluble egg antigen.

We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

Much effort has gone into finding peptides that bind potentially useful nanoparticles, but relatively little effort has focused on the scaffolds that organize these peptides into useful nanostructures. Chaperonins are protein complexes with 14-18 protein subunits that self-assemble into double-ring complexes and function as scaffolds for peptides or amino acids that bind metallic and semiconductor quantum dots. The utility of chaperonins as scaffolds depends on their structure and their ability to self-assemble into double-rings and higher-order structures, such as filaments and two-dimensional arrays. To better understand the structure of chaperonins, we constructed a model of a group II chaperonin and, based on this model, genetically constructed five mutant subunits with significant deletions. We expressed these mutants as recombinant proteins and observed by native polyacrylamide gel electrophoresis (PAGE) and transmission electron microscopy (TEM) that they all self-assembled into double rings. Our model predicted and TEM confirmed that these deletions did not significantly change the 17 nm diameter of the wild-type double rings, but decreased their height and opened their central cavities. Four of the five mutants formed higher-order structures: chains of rings, bundles of chains or filaments, and two-dimensional arrays, which we suggest can be useful nanostructures.

SUMMARY Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular-lesion. Sleep was increased using three independent strategies: activating the dorsal Fan Shaped Body (FB), increasing the expression of Fatty acid binding protein (dFabp) or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or Long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using Aversive Phototaxic Suppression (APS) and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer’s disease. Together these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggests that increasing sleep may benefit patients with certain neurological disorders. PMID:25913403

Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein–Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant. PMID:19259066

Activating mutations in Neuroblastoma RAS viral oncogene homolog (NRAS) are found in 15-30% of melanomas and are associated with a poor prognosis. Although MAP kinase kinase (MEK) inhibitors used as single agents showed a limited clinical benefit in patients with NRAS-mutant melanoma due to their rather cytostatic effect and high toxicity, their combination with other inhibitors of pathways known to cooperate with MEK inhibition may maximise their antitumour activity. Similarly, in a context where p53 is largely inactivated in melanoma, hyperexpression of Microphthalmia associated transcription factor (MITF) and its downstream anti-apoptotic targets may be the cause of the restraint cytotoxic effects of MEK inhibitors. Indeed, drug combinations targeting both mutant BRAF and MITF or one of its important targets Bcl-2 were effective in mutant BRAF melanoma but had no effect on acquired resistance. Therefore, we aimed to further investigate the downstream MITF targets that can explain this anti-apoptotic effect and to evaluate in parallel the effect of p53 reactivation on the promotion of apoptosis under MEK inhibition in a panel of (Q61)NRAS-mutant melanoma cells. First, we showed that MEK inhibition (pimasertib) led to a significant inhibition of cell proliferation but with a limited effect on apoptosis that could be explained by the systematic MITF upregulation. Mimicking the MITF effect via cyclic adenosine monophosphate activation conferred resistance to MEK inhibition and upregulated Bcl-2 expression. In addition, acquired resistance to MEK inhibition was associated with a strong activation of the anti-apoptotic signalling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knockout using CRISPR/Cas9 system annihilated the acquired resistance and restored the sensitivity of NRAS-mutant melanoma cells to MEK inhibition. Strikingly and similarly, direct p53 reactivation (PRIMA-1(Met), APR-246) also broke resistance and synergised with

Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer.

For the construction of strains with full probiotics function in intestines, deoxycholate resistant mutants were isolated from Bacillus subtilis natto. The partial characterization of the mutants was carried out and described.

Fabry disease is an X-linked inherited lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3) due to α-galactosidase A (α-Gal A) deficiency. Fabry patients frequently report of anxiety, depression, and impaired cognitive function. We characterized affective and cognitive phenotype of male mice with α-Gal Adeficiency (Fabry KO) and compared results with those of age-matched male wildtype (WT) littermates. Young (3 months) and old (≥ 18 months) mice were tested in the naïve state and after i.pl. injection of complete Freund`s adjuvant (CFA) as an inflammatory pain model. We used the elevated plus maze (EPM), the light-dark box (LDB) and the open field test (OF) to investigate anxiety-like behavior. The forced swim test (FST) and Morris water maze (MWM) were applied to assess depressive-like and learning behavior. The EPM test revealed no intergroup difference for anxiety-like behavior in naïve young and old Fabry KO mice compared to WT littermates, except for longer time spent in open arms of the EPM for young WT mice compared to young Fabry KO mice (p<0.05). After CFA injection, young Fabry KO mice showed increased anxiety-like behavior compared to young WT littermates (p<0.05) and naïve young Fabry KO mice (p<0.05) in the EPM as reflected by shorter time spent in EPM open arms. There were no relevant differences in the LDB and the OF test, except for longer time spent in the center zone of the OF by young WT mice compared to young Fabry KO mice (p<0.05). Complementary to this, depression-like and learning behavior were not different between genotypes and age-groups, except for the expectedly lower memory performance in older age-groups compared to young mice. Our results indicate that genetic influences on affective and cognitive symptoms in FD may be of subordinate relevance, drawing attention to potential influences of environmental and epigenetic factors.

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1Adeficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1Adeficiency resulted in fulminant pancolitis with early expansion of CD11b+ myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1β activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b+Ly6C+-derived IL-1β production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b+ myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1β, consistent with pyroptosis. Inhibition of reactive oxygen species–mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis. PMID:23636052

Fabry disease is an X-linked inherited lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3) due to α-galactosidase A (α-Gal A) deficiency. Fabry patients frequently report of anxiety, depression, and impaired cognitive function. We characterized affective and cognitive phenotype of male mice with α-Gal Adeficiency (Fabry KO) and compared results with those of age-matched male wildtype (WT) littermates. Young (3 months) and old (≥ 18 months) mice were tested in the naïve state and after i.pl. injection of complete Freund`s adjuvant (CFA) as an inflammatory pain model. We used the elevated plus maze (EPM), the light-dark box (LDB) and the open field test (OF) to investigate anxiety-like behavior. The forced swim test (FST) and Morris water maze (MWM) were applied to assess depressive-like and learning behavior. The EPM test revealed no intergroup difference for anxiety-like behavior in naïve young and old Fabry KO mice compared to WT littermates, except for longer time spent in open arms of the EPM for young WT mice compared to young Fabry KO mice (p<0.05). After CFA injection, young Fabry KO mice showed increased anxiety-like behavior compared to young WT littermates (p<0.05) and naïve young Fabry KO mice (p<0.05) in the EPM as reflected by shorter time spent in EPM open arms. There were no relevant differences in the LDB and the OF test, except for longer time spent in the center zone of the OF by young WT mice compared to young Fabry KO mice (p<0.05). Complementary to this, depression-like and learning behavior were not different between genotypes and age-groups, except for the expectedly lower memory performance in older age-groups compared to young mice. Our results indicate that genetic influences on affective and cognitive symptoms in FD may be of subordinate relevance, drawing attention to potential influences of environmental and epigenetic factors. PMID:28662189

A mutant of Mycobacterium smegmatis unable to use the dipeptide carnosine (β-alanyl-l-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on β-Ala and/or l-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium. PMID:9852030

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed. PMID:4992372

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficientmutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human β-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrAdeficientmutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions.

Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes ofmore » mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.

Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit, at Lubbock, Texas. The two mutants, erl1-1 and erl1-2, were isol...

SUMMARY Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn’s disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyper-immune phenotype and protective effects are lost in Atg16L1/Nod2 double mutant mice. These results reveal an immuno-suppressive function of Atg16L1, and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections. PMID:23954160

The development of root hairs serves as an excellent model to study cell growth using both cytological and genetic approaches. In the past, we have characterized LRX1, an extracellular protein of Arabidopsis consisting of an LRR-domain and a structural extensin domain. LRX1 is specifically expressed in root hairs and lrx1 mutants show severe deficiencies in root hair development. In this work, we describe the characterization of enl (enhancer of lrx1) mutants that were isolated in a visual screen of an ethylmethanesulfonate -mutagenized lrx1 line for plants exhibiting an enhanced lrx1 phenotype. Four recessive enl mutants were analyzed, three of which define new genetic loci involved in root hair development. The mutations at the enl loci and lrx1 result in additive phenotypes in enl/lrx1 double mutants. One enl mutant is affected in the ACTIN2 gene and encodes a protein with a 22 amino acid deletion at the C-terminus. The comparison of molecular and phenotypic data of different actin2 alleles suggests that the truncated ACTIN2 protein is still partially functional.

This review article points out that bananas are an important food for many people in the world. Thus, banana cultivars rich in provitamin A carotenoids may offer a potential food source for alleviating vitamin Adeficiency, particularly in developing countries. Many factors are associated with the presently known food sources of vitamin A that limit their effectiveness in improving vitamin A status. Acceptable carotenoid-rich banana cultivars have been identified in Micronesia, and some carotenoid-rich bananas have been identified elsewhere. Bananas are an ideal food for young children and families for many regions of the world, because of their sweetness, texture, portion size, familiarity, availability, convenience, versatility, and cost. Foods containing high levels of carotenoids have been shown to protect against chronic disease, including certain cancers, cardiovascular disease, and diabetes. Because the coloration of the edible flesh of the banana appears to be a good indicator of likely carotenoid content, it may be possible to develop a simple method for selecting carotenoid-rich banana cultivars in the community. Research is needed on the identification of carotenoid-rich cultivars, targeting those areas of the world where bananas are a major staple food; investigating factors affecting production, consumption, and acceptability; and determining the impact that carotenoid-rich bananas may have on improving vitamin A status. Based on these results, interventions should be undertaken for initiating or increasing homestead and commercial production.

A total of 240 children were examined for vitamin Adeficiency in a village in Hararge region of Ethiopia. Night blindness, Bitot's spots, corneal xerosis, corneal ulceration and corneal scars were observed in 69, 16, 2, 15 and 14 children, respectively, based on the most severe eye signs. Blood was collected from 76 children with eye signs and 9 other children selected at random. The concentration of retinol (vitamin A) in serum was < 0.35 mumol/l in 30.2% of children and the median serum retinol-binding protein, iron, transferrin saturation and ferritin levels were low while the parameters of iodine status, total triiodothyronine, total thyroxine and thyrotropin, were all within the normal range. Levels of IgG and IgM were elevated in 78.8% and 82.4% children, respectively, while C-reactive protein levels were elevated in 42.4% of children. There was a higher prevalence of wasting (33%) than stunting (10%) with an additional 8% of children being both stunted and wasted. In the 2 years prior to the study, there were 74 deaths of which 17 were reported to be associated with ruptured corneas. The community had been dependent on relief food aid for the previous 6 years.

The decreased dietary diversity wrought from the adoption of the settled, agrarian system to replace the hunter-gather and pastoralist lifestyles assured a stable supply of protein and calories from grains and tubers while creating a vulnerability for humans to suffer micronutrient deficiencies. The vitamin A from animal tissue is more bioavailable to humans than the provitamin A in the matrix of green plants. Provitamin A carotenes achieve a dietary vitamin A efficacy nearly equivalent to that of the preformed vitamin only in the context of an oily matrix. The homeostatic regulation of carotene bioconversion by the intestine, moreover, prevents any excess toxic accumulation of vitamin A from provitamin A sources. The efficacy and safety of the palm fruit (genus Elaeis) as a source of vitamin A, in addition to its cultural recognition as a food, are more consistent with the gentler concept of "alleviation" of the public health problem of hypovitaminosis A, then the more aggressive, medical model of "eradication" with its greater potential for risk and collateral damage. The palm fruit and its derivatives achieve new opportunities for creative contribution and sustained use in formats of supplementation (prophylactic in children and women, for lactation), food-to-food fortification (in bakery goods and snacks, as condiments), and even in food diversification strategies. Experience in India, South Africa, and Guatemala begins to define and delineate the opportunities and limitations for the palm fruit to contribute to the alleviation of endemic vitamin Adeficiency.

The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45Adeficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride. PMID:27820820

Micronutrient deficiency is an unquestionable public health problem, specially anemia and vitamin Adeficiency (VAD). This is due to the collective dimension of these carencies, which reflects on morbimortality rates in the maternal and infant group. to evaluate the impact of a proposal for prenatal nutritional assistance, comparing the prevalence of anemia and VAD, in pre-intervention (GI) and intervention (GII) groups. this is a prospective intervention study in a cohort of pregnant women. The GI group was made up of 225 the GII group of 208 pregnant adults and their respective newborns, attended a Public Maternity Ward in Rio de Janeiro, Brazil. Concentration of hemoglobin was used to diagnose anemia and a standardized interview to diagnose night blindness (XN). after adjusting for confounding variables, through logistic regression, the protective effect of intervention at the onset of anemia (OR = 0.420; IC 95% = 0.251-0.702), with a significant reduction in prevalence, of 28.4% in the GI to 16.8% in the GII, also observed at the onset of XN (OR = 0.377; IC95% = 0.187- 0.759), with a reduction in prevalence of 18.7 % in the GI to 6.2% in the GII. Nutritional intervention has a beneficial effect on maternal health, reducing nutritional deficiencies most prevalent during pregnancy and the impact of these on the obstetric ailment.

The β-carotene content of fresh and dried mangoes commonly consumed in Kenya was evaluated and converted to retinol equivalent (RE). Mango fruits of varieties Ngowe, Apple, and Tommy Atkins were harvested at mature green, partially ripe, and ripe stages and their β-carotene content analyzed. The stability of β-carotene in sun dried mangoes was also studied over 6 months under usual marketing conditions used in Kenya. The effect of using simple pretreatment methods prior to drying of mango slices on retention of β-carotene was as well evaluated. In amounts acceptable to children and women, fresh and dried mangoes can supply 50% or more of the daily required retinol equivalent for children and women. Stage of ripeness, variety, postharvest holding temperature, method of drying, and storage time of dried mango slices affected β-carotene content and consequently vitamin A value of the fruits. Apple variety grown in Machakos had the highest β-carotene. It exceeded the daily RE requirements by 11.8% and 21.5% for women and children respectively. Fresh or dried mangoes are a significant provitamin A source and should be included in food-based approaches aiming to reduce vitamin Adeficiency.

Francisella tularensis, the causative agent of tularemia, is most deadly in the pneumonic form; therefore, mucosal immunity is an important first line of defense against this pathogen. We have now evaluated the lethality of primary F. tularensis live vaccine strain (LVS) pulmonary infection in mice that are defective in IgA (IgA(-/-) mice), the predominant mucosal Ig isotype. The results showed that IgA(-/-) mice were more susceptible than IgA(+/+) mice to intranasal F. tularensis LVS infection, despite developing higher levels of LVS-specific total, IgG, and IgM antibodies in the bronchoalveolar lavage specimens following infection. In addition, the absence of IgA resulted in a significant increase in bacterial loads and reduced survival. Interestingly, IgA(-/-) mice had lower pulmonary gamma interferon (IFN-γ) levels and decreased numbers of IFN-γ-secreting CD4(+) and CD8(+) T cells in the lung on day 9 postinfection compared to IgA(+/+) mice. Furthermore, IgA(-/-) mice displayed reduced interleukin 12 (IL-12) levels at early time points, and supplementing IgA(-/-) mice with IL-12 prior to LVS challenge induced IFN-γ production by NK cells and rescued them from mortality. Thus, IgA(-/-) mice are highly susceptible to primary pulmonary LVS infections not only because of IgAdeficiency but also because of reduced IFN-γ responses.

Genetic variations in the cdk5 regulator associated protein 1-like 1 (cdkal1) gene have been identified in whole genome association studies as a risk factor for the development of type 2 diabetes (T2D). A recent study showed that Cdkal1 was a mammalian methythiotransferase, which specifically synthesizes 2-methylthio-N (6)-threonylcarbamoyladenosine (ms (2)t (6)A) at position 37 of tRNA(lys)(UUU). The ms (2)t (6)A modification in tRNA(lys)(UUU) was important for the accurate decoding of its cognate codon. In pancreatic β-cell-specific Cdkal1 knockout (Cdkal1 KO) mice, adeficiency of ms (2)t (6)A caused the mistranslation of a Lys codon in proinsulin, resulting in improper processing. The mice showed a decrease in insulin secretion and glucose intolerance. In addition, the mistranslation contributed to the expression of the endoplasmic reticulum (ER) stress response in Cdkal1-deficient β-cells. Furthermore, Cdkal1 KO mice were hypersensitive to high-fat diet-induced glucose intolerance, as well as the ER stress response. These findings might potentially explain the molecular pathogenesis of T2D in patients carrying Cdkal1 variations.

B cell CLL/lymphoma 11A (BCL11A) is a transcription factor and regulator of hemoglobin switching that has emerged as a promising therapeutic target for sickle cell disease and thalassemia. In the hematopoietic system, BCL11A is required for B lymphopoiesis, yet its role in other hematopoietic cells, especially hematopoietic stem cells (HSCs) remains elusive. The extensive expression of BCL11A in hematopoiesis implicates context-dependent roles, highlighting the importance of fully characterizing its function as part of ongoing efforts for stem cell therapy and regenerative medicine. Here, we demonstrate that BCL11A is indispensable for normal HSC function. Bcl11adeficiency results in HSC defects, typically observed in the aging hematopoietic system. We find that downregulation of cyclin-dependent kinase 6 (Cdk6), and the ensuing cell-cycle delay, correlate with HSC dysfunction. Our studies define a mechanism for BCL11A in regulation of HSC function and have important implications for the design of therapeutic approaches to targeting BCL11A.

Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

In 1993, the Department of Health of the Federated States of Micronesia (FSM) conducted a population-based stratified random survey among 355 children aged 24-48 months in Pohnpei, one of the four FSM States. The objective was to determine the prevalence, and explore risk factors for vitamin Adeficiency (VAD). Trained field workers collected data from a range of demographic, dietary and socioeconomic variables related to the children. The serum retinol concentration was 19.4 +/- 7.5 microg/dl (mean +/- SD), and the VAD prevalence (serum retinol <20 microg/dl) 53.1 per cent. The significant independent risk factors, determined by logistic regression, were: mother's work at home, sibling <2 years older, rural household located on the main island, early weaning, and child anemia, controlling for pipe water and electricity in the household. If compared with a reference of apparently healthy children of similar age in the USA, the distribution of serum retinol among young Pohnpei children was shifted entirely to low levels. We conclude that eliminating the pervasive VAD problem in Pohnpei would require a multi-pronged tactical approach that combines dietary improvement strategies with the ongoing supplementation effort.

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI: http://dx.doi.org/10.7554/eLife.00444.001 PMID:23580231

Vitamin Adeficiency (VAD) remains an important problem in the developing world where environmental air pollution is increasing. Because the coincidence of these factors could increase the prevalence of asthma in these regions, airway reactivity to methacholine, capsaicin, and sulfur dioxide was studied in VAD rats. The impedance to a small volume oscillation was analyzed to determine airway resistance (Raw) and lung elastance (H). VAD rats demonstrated larger increases in Raw and H after the administration of methacholine, and in Raw after administration of capsaicin or sulfur-dioxide. The muscarinic receptor-2 (M2R) selective antagonist AFDX-116 enhanced the effect of capsaicin in vitamin A sufficient (VAS) but not VAD rats and retinoic acid administration partially restored this enhancement. These data indicate that diminished auto-inhibitory muscarinic receptor-2 (M2R) function contributed to this increased airway responsiveness to pulmonary C-fiber stimulation in VAD rats. If children with VAD also have diminished M2R function, they may be more prone to develop asthma, particularly in the presence of environmental co-factors such as sulfur dioxide. PMID:17321224

The Th1/ Th2 paradigm has become an important issue in the pathogenesis of asthma, characterized by normal Th-1 and elevated Th-2 cytokine expression, resulting in a Th2 predominance. Vitamin Adeficiency (VAD) produces a significant Th1 bias, while high-level dietary vitamin A supplementation promo...

Background Screening for IgAdeficiency in patients with coeliac disease is essential because of the increased incidence of IgAdeficiency associated with the disease, which usually relies on the estimation of IgA levels in each case. Aim To devise a method of excluding IgAdeficiency without measuring total serum IgA in each case. Materials and methods The optical density readings on enzyme‐linked immunosorbent assay (ELISA) of 608 routine samples received for tissue transglutaminase (TTG) antibody testing for coeliac disease were compared with their total IgA concentrations. Dilution experiments were also carried out to ensure linear relationships between optical density on ELISA and IgA concentrations and to compare the sensitivities for TTG and endomysium antibodies in TTG‐positive samples. Results and discussion A clear relationship was shown between total IgA concentration and TTG optical density readings by ELISA. To ensure a positive TTG result if antibodies are present, it was possible to recommend an optical density level above which all samples have sufficient IgA. Samples with optical density <0.05 should be investigated further by estimating total IgA and, if low, samples should be subjected to immunofluorescence microscopy testing for IgA and IgG endomysium antibodies. Conclusions An easier, more cost‐effective and practical way of excluding IgAdeficiency in the investigation on coeliac disease is reported. PMID:16489174

The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 x SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing beta-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4.7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 beta-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

The anticancer drug 5-fluorouracil (5-FU) and its metabolite 5-fluoro-2'-deoxyuridine (FUdR) inhibit thymidylate synthase and induce uracil bases in DNA. FUdR is commonly used for inhibiting fertility when measuring the lifespan of the nematode Caenorhabditis elegans. However, it is not known whether DNA damage induced by FUdR affects lifespan. EXO-3 is an apurinic/apyrimidinic endonuclease in C. elegans, and we reported previously that deletion of the exo-3 gene causes reproductive abnormalities and decreased lifespan. In this study, we found that FUdR extended the lifespan of exo-3 mutants. We measured the lifespan of multiple germline mutants to examine whether this lifespan extension effect was dependent on fertility. In the presence of a fem-1 mutation, which causes adeficiency in sperm production, FUdR did not extend the lifespan of the exo-3 mutant. In glp-1 mutants, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR did not extend its lifespan. These results suggest that the lifespan extension effect of FUdR depends on fertility and the presence of gonads. fem-3 mutants, which do not produce oocytes, had increased lifespan in the presence of FUdR, independent of the exo-3 mutation. It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR. From these results, we suggest that FUdR affects the lifespan of C. elegans in two ways: by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan.

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.

Hungry fruit flies can be trained by exposing them to two chemical odorants, one paired with the opportunity to feed on 1 M sucrose. On later testing, when given a choice between odorants the flies migrate specifically toward the sucrose-paired odor. This appetitively reinforced learning by the flies is similar in strength and character to previously demonstrated negatively reinforced learning, but it differs in several properties. Both memory consolidation and memory decay proceed relatively slowly after training with sucrose reward. Consolidation of learned information into anesthesia-resistant long-term memory requires about 100 min after training with sucrose compared to about 30 min after training with electric shock. Memory in wild-type flies persists for 24 hr after training with sucrose compared to 4-6 hr after training with electric shock. Memory in amnesiac mutants appears to be similarly lengthened, from 1 hr to 6 hr, by substituting sucrose reward for shock punishment. Two other mutants, dunce and rutabaga, which were isolated because they failed to learn the shock-avoidance task, learn normally in response to sucrose reward but forget rapidly afterward. One mutant, turnip, does not learn in either paradigm. Reward and punishment can be combined in olfactory discrimination training by pairing one odor to sucrose and the other to electric shock. In this situation, the expression of learning is approximately the sum of that obtained by using either reinforcement alone. After such training, memory decays at two distinct rates, each characteristic of one type of reinforcement. PMID:6572401

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin Adeficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin Adeficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine. PMID:8359917

Aging is an important biological process that affects all creatures. For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact. We can therefore expect that models to study mechanisms of the aging process will always attract much interest. Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies. However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists. A brief review of current models is presented.

examined the ability of each E1A mutant to induce p53. Cells expressing full-length E1A dis - played a 20- to 30-fold increase in steady-state p53...retinoblastoma (Rb) protein, and inac- tivation of both is essential for viral transformation (Lane and Crawford 1979; Linzer and Levine 1979; De- Caprio et al...Western blotting. Despite the fact that wild-type MEFs expressing El A dis - played an -10-fold increase in p53 and Mdm2 levels as compared to their Ai?F

Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

A cross-sectional study was conducted to assess the prevalence and correlates of prenatal vitamin Adeficiency (VAD) in rural Sidama, Southern Ethiopia. Seven hundred randomly-selected pregnant women took part in the study. Serum retinol concentration was determined using high-performance liquid chromatography. Data were analyzed by logistic and linear regression. Interpretation of data was made using adjusted odds ratio (AOR) and adjusted linear regression coefficient. The prevalence of VAD (serum retinol < 0.7 micromol/L) was 37.9%. Advanced gestational age and elevated C-reactive protein (CRP > or = 5 mg/dL) were negatively associated with retinol concentration (p < 0.05). The odds of VAD was significantly higher among the women with no education and those devoid of self-income. Women aged 35-49 years had 2.23 (95% CI 1.31-3.81) times higher odds compared to those aged 15-24 years. The lower the dietary diversity score in the preceding day of the survey, the higher were the odds of VAD. With reference to nulliparas, grand multiparas had 1.92 (95% CI 1.02-3.64) times increased odds of VAD. VAD and zinc deficiency (serum zinc <8.6 pmol/L during the first trimester, or <7.6 pmol/L during the second or third trimester) were significantly associated with AOR of 1.80 (95% CI 1.28-2.53). VAD has major public-health significance in the area. Accordingly, it should be combated through enhancement of diet diversity, birth control, and socioeconomic empowerment of women.

To investigate the prevalence of vitamin Adeficiency (VAD) among pregnant women in rural Bangladesh, and examine the relationship between various factors and vitamin A status. Community Nutrition Promoter (CNP) centres in Kapasia sub-district of Gazipur district, Bangladesh. A cross-sectional study. Two hundred women, aged 18-39 years, in their second or third trimester of pregnancy were selected from seventeen CNP centres in four unions of Kapasia sub-district where they usually visit for antenatal care. Various socio-economic, personal and pregnancy-related information, dietary intake of vitamin A and mid-upper arm circumference (MUAC) data were collected. Serum retinol (vitamin A) concentration was determined. More than half (51 %) of the pregnant women had low vitamin A status (serum retinol <1.05 micromol/l) with 18.5 % having VAD (serum retinol <0.70 micromol/l). Fifty-three per cent of the women's vitamin A intake was less than the recommended dietary allowance. By multiple regression analysis, MUAC, per-capita expenditure on food and wealth index were found to have significant independent positive relationship with serum retinol concentration, while gestational age of the pregnant women had a negative relationship. The overall F-ratio (10.3) was highly significant (P = 0.0001), the adjusted R2 was 0.18 (multiple R = 0.45). VAD is highly prevalent among rural pregnant women in Bangladesh. Gestational age, nutritional status, per-capita expenditure on food and wealth index appear to be important in influencing the vitamin A status of these women. An appropriate intervention is warranted in order to improve the vitamin A status.

In developed countries, food fortification has proven an effective and low-cost way to increase the micronutrient supply and reduce the consequences of micronutrient deficiencies. It has been rarely used in the developing world, but general conclusions can be drawn. The biological efficacy, but not the effectiveness, of fortifying oil and hydrogenated oil products as well as cereal flours and meals with vitamin A has been shown. Sugar has been fortified with vitamin A in Central American countries for years, and biological efficacy and program effectiveness are well established. Efficacy of fortifying monosodium glutamate with vitamin A was demonstrated but a program has not been established. Fortification with vitamin A in the developing world should satisfy certain elements for success. a) A potential food matrix (a food regularly consumed, produced by a few centralized factories, without sensorial changes compared with the nonfortified equivalent, and nutrient remains bioavailable and in a sufficient amount) is required. b) Fortified foods should provide at least 15% of the recommended daily intakes for the target group (e.g., individuals consuming the lowest amount of the fortified food). c) Voluntary fortification of processed foods should be regulated to prevent excessive consumption of vitamin A. d) Neighboring countries should harmonize technical standards, facilitate compliance and minimize conflicts over global trade laws. e) A practical monitoring system should be instituted. f) Social marketing activities should be permanent and aimed at industry, government and consumers. g) Food fortification should be combined with other strategies (e.g., supplementation) to reach those not adequately covered by fortification alone. Infants and small children, whose dietary habits differ from those of adults, require special attention. Fortification of food commodities is a very attractive and economic way to prevent and control vitamin Adeficiency. Effective food

Vitamin Adeficiency (VAD) has been known to exist in Latin America and the Caribbean since the mid-1960s; however, except for pioneering work by the Institute of Nutrition of Central America and Panama/Pan American Health Organization on sugar fortification in Central America, there was little interest in controlling it because of the low frequency of clinical findings. More recently, implications of the effect of subclinical VAD on child health and survival has generated increased interest in assessing the problem and a greater commitment to controlling it. The information available by mid-1997 on the magnitude of VAD in countries of the Region was extensively reviewed. Internationally accepted methods and cutoff points for prevalence estimations were used to compile information from relevant dietary, biochemical, and clinical studies carried out between 1985 and 1997 in samples of at least 100 individuals. VAD in the Region of Latin America and the Caribbean is mostly subclinical. The national prevalence of subclinical VAD (serum retinol < 20 micrograms/dl) in children under 5 years of age ranges between 6% in Panama and 36% in El Salvador. The problem is severe in five countries, moderate in six, and mild in four. There are no recent data from Chile, Haiti, Paraguay, Uruguay, Venezuela, and the English-speaking Caribbean. The population affected amounts to about 14.5 million children under 5 years of age (25% of that age group). Schoolchildren and adult women may also have significant VAD. Actions currently implemented to control VAD include (a) universal or targeted supplementation, with sustained high coverage rates through national immunization days in some countries; (b) sugar fortification, which is well established in El Salvador, Guatemala, and Honduras (a significant effect has been documented in Guatemala and Honduras) and is under negotiation in Bolivia, Colombia, Costa Rica (to be resumed), Ecuador, Nicaragua, and Peru; and (c) limited dietary

Vitamin A, a fat-soluble, heat-stable nutrient (retinol) derived from animal sources and certain fruits and vegetables, forms the basic component of retinal pigments and plays a vital role in optimal health, growth, and development. Vitamin Adeficiency (VAD) (serum retinol < or = 20 microg/dL [< or = 0.7 micromol/L] for subclinical VAD) can substantially increase the risk for childhood mortality from infectious and noninfectious causes. VAD impairs the mobilization and transport of iron and is usually associated with anemia and reduced growth. VAD is a major public health problem in parts of Africa, Asia, Latin America, and the Western Pacific. In Chuuk and Pohnpei, two of the four Federated States of Micronesia (FSM) (2000 population: 107,008), nutrition surveys during the early 1990s documented VAD prevalences among the highest in the world (CDC, unpublished data, 1991; U.S. Public Health Service, unpublished data, 1994). In response to these findings, FSM health authorities, with support of the United Nations Children's Fund (UNICEF), began distributing vitamin A supplements in 1993 and 1998 in Chuuk and Pohnpei, respectively. In November 1999, FSM requested assistance from CDC in VAD assessment surveys of children in Kosrae and Yap, the other two FSM states. This report summarizes levels of serum retinol and prevalence of VAD and other indicators of nutritional status among children aged 24-59 months in Kosrae and Yap. The findings indicated low serum retinol levels and high VAD prevalences but no substantial stunting or wasting. A comprehensive, long-term national strategy is needed in FSM to promote sustained improvement in vitamin A status.

Vitamin Adeficiency (VAD) has been recognized as a public-health issue in developing countries. Economic constraints, sociocultural limitations, insufficient dietary intake, and poor absorption leading to depleted vitamin A stores in the body have been regarded as potential determinants of the prevalence of VAD in South Asian developing countries. VAD is exacerbated by lack of education, poor sanitation, absence of new legislation and enforcement of existing food laws, and week monitoring and surveillance system. Several recent estimates confirmed higher morbidly and mortality rate among children and pregnant and non-pregnant women of childbearing age. Xerophthalmia is the leading cause of preventable childhood blindness with its earliest manifestations as night blindness and Bitot's spots, followed by blinding keratomalacia, all of which are the ocular manifestations of VAD. Children need additional vitamin A because they do not consume enough in their normal diet. There are three general ways for improving vitamin A status: supplementation, fortification, and dietary diversification. These approaches have not solved the problem in South Asian countries to the desired extent because of poor governmental support and supervision of vitamin A supplementation twice a year. An extensive review of the extant literature was carried out, and the data under various sections were identified by using a computerized bibliographic search via PubMed, Web of Science, and Google Scholar. All abstracts and full-text articles were examined, and the most relevant articles were selected for screening and inclusion in this review. Conclusively, high prevalence of VAD in South Asian developing countries leads to increased morbidity and mortality among infants, children, and pregnant women. Therefore, stern efforts are needed to address this issue of public-health significance at local and international level in lower- and middle-income countries of South Asia. PMID:24592582

Vitamin Adeficiency (VAD) has been recognized as a public-health issue in developing countries. Economic constraints, sociocultural limitations, insufficient dietary intake, and poor absorption leading to depleted vitamin A stores in the body have been regarded as potential determinants of the prevalence of VAD in South Asian developing countries. VAD is exacerbated by lack of education, poor sanitation, absence of new legislation and enforcement of existing food laws, and week monitoring and surveillance system. Several recent estimates confirmed higher morbidly and mortality rate among children and pregnant and non-pregnant women of childbearing age. Xerophthalmia is the leading cause of preventable childhood blindness with its earliest manifestations as night blindness and Bitot's spots, followed by blinding keratomalacia, all of which are the ocular manifestations of VAD. Children need additional vitamin A because they do not consume enough in their normal diet. There are three general ways for improving vitamin A status: supplementation, fortification, and dietary diversification. These approaches have not solved the problem in South Asian countries to the desired extent because of poor governmental support and supervision of vitamin A supplementation twice a year. An extensive review of the extant literature was carried out, and the data under various sections were identified by using a computerized bibliographic search via PubMed, Web of Science, and Google Scholar. All abstracts and full-text articles were examined, and the most relevant articles were selected for screening and inclusion in this review. Conclusively, high prevalence of VAD in South Asian developing countries leads to increased morbidity and mortality among infants, children, and pregnant women. Therefore, stem efforts are needed to address this issue of public-health significance at local and international level in lower- and middle-income countries of South Asia.

At least in mammals, retinoic acid is a pivotal factor in maintaining the functionality of the testis, in particular, for the progression of germ cells from mitosis to meiosis. Removal of dietary vitamin A or a targeted deletion of retinoic acid receptor alpha gene (Rara), the receptor for retinoic acid, in mice, led to testicular degeneration by a dramatic loss of germ cells and a loss of control of the spermatogenic cycle. The germ cells that remained in the vitamin Adeficient (VAD) rat testis were spermatogonia and a few preleptotene spermatocytes. Spermatogenesis can be reinitiated by injection of VAD rats with retinol, the metabolic precursor of retinoic acid, but to date, the functions of retinoic acid in the testis remain elusive. We have applied DNA microarray technology to investigate the time-dependent transcriptome changes that occur 4 to 24 h after retinol replenishment in the VAD rat testis. The retinol-regulated gene expression occurred both in germ cells and Sertoli cells. Bioinformatic analyses revealed time-dependent clusters of genes and canonical pathways that may have critical functions for proper progression through spermatogenesis. In particular, gene clusters that emerged dealt with: (1) cholesterol and oxysterol homeostasis, (2) the regulation of steroidogenesis, (3) glycerophospholipid metabolism, (4) the regulation of acute inflammation, (5) the regulation of the cell cycle including ubiquitin-mediated degradation of cell cycle proteins and control of centrosome and genome integrity, and (6) the control of membrane scaffolding proteins that can integrate multiple small GTPase signals within a cell. These results provide insights into the potential role of retinoic acid in the testis. PMID:19886770

Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30–34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility. PMID:16888336

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and "second positive" phototropism. However, while the amplitude for "first positive" phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in "first positive" phototropism are shifted to higher fluence by a factor of 20-30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 micromole m-2 to 2700 micromoles m-2, but shows normal gravitropism. Strain JK345 shows no "first positive" phototropism, and reduced gravitropism and "second positive" phototropism. Strain JK229 shows no measurable "first positive" phototropism, but normal gravitropism and "second positive" phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. "first positive" and "second positive" phototropism contain at least one common element; and 3. "first positive" phototropism can be substantially altered without any apparent alteration of "second positive" phototropism.

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

Revertible, spontaneous Nif- mutants of Rhodopseudomonas capsulata have been shown to accumulate in cultures growing photosynthetically with an amino acid as the nitrogen source such that H2 is maximally produced. The majority of such strains carry mutations which are clustered in a short region of the chromosome, probably representing one or two genes. Because this cluster includes temperature-sensitive mutations, it is also likely that it identifies the structural gene of a polypeptide. The phenotypic characterization of these spontaneous mutants showed (i) an inability to grow with N2 as the nitrogen source, no measurable nitrogenase activity, a reduction or absence of the three polypeptides of the MoFe and Fe proteins of the nitrogenase complex, a faster growth rate on glutamate as the nitrogen source under saturating light, and frequently a small increase in glutamine synthetase activity relative to that of the wild type when grown with glutamate as the nitrogen source. Alterations in other ammonium-assimilatory enzyme activities were not observed. Taken together, these properties suggest that the mutations have affected a regulatory protein necessary for nitrogen fixation. Images PMID:6146598

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and "second positive" phototropism. However, while the amplitude for "first positive" phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in "first positive" phototropism are shifted to higher fluence by a factor of 20-30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 micromole m-2 to 2700 micromoles m-2, but shows normal gravitropism. Strain JK345 shows no "first positive" phototropism, and reduced gravitropism and "second positive" phototropism. Strain JK229 shows no measurable "first positive" phototropism, but normal gravitropism and "second positive" phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. "first positive" and "second positive" phototropism contain at least one common element; and 3. "first positive" phototropism can be substantially altered without any apparent alteration of "second positive" phototropism.

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These “multiples” appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts. PMID:17687667

Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non- coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2- terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over- expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis. PMID:8335685

Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic. PMID:15199170

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These "multiples" appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts.

Mutants of Salmonella typhimurium containing 1 to 2% of wild-type ribonuclease I activity were isolated. The rns mutation had no effect on the polarity of mutations in the S. typhimurium histidine operon. Even in the presence of an rns mutation, it was not possible to obtain strong suppressors of the polarity of two polar mutations in the his operon. PMID:4347966

The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1(R132H) mutation destabilizes an IDH1 "regulatory segment," which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity. PMID:12024028

A planting of 3,919 M1 kernels from normal ears crossed by EMS-treated pollen produced 3,461 M1 plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive. PMID

A planting of 3,919 M(1) kernels from normal ears crossed by EMS-treated pollen produced 3,461 M(1) plants and 3,172 selfed ears. These plants yielded 2,477 (72%) total heritable changes; the selfed ears yielded 2,457 (78%) recessive mutants, including 855 (27%) recessive kernel mutants and 8 (0.23%) viable dominant mutants. The ratio of recessive to dominant mutants was 201:1. The average mutation frequency for four known loci was three per 3,172 genomes analyzed. The estimated total number of loci mutated was 535 and the estimated number of kernel mutant loci mutated was 285. Among the 855 kernel mutants, 432 had a nonviable embryo, and 59 germinated but had a lethal seedling. A sample of 194 of the latter two types was tested for heritability, lethality, chromosome arm location and endosperm-embryo interaction between mutant and nonmutant tissues in special hyper-hypoploid combinations produced by manipulation of B-A translocations. The selected 194 mutants were characterized and catalogued according to endosperm phenotype and investigated to determine their effects on the morphology and development of the associated embryo. The possibility of rescuing some of the lethal mutants by covering the mutant embryo with a normal endosperm was investigated. Ninety of these 194 mutants were located on 17 of the 18 chromosome arms tested. Nineteen of the located mutants were examined to determine the effect of having a normal embryo in the same kernel with a mutant endosperm, and vice versa, as compared to the expression observed in kernels with both embryo and endosperm in a mutant condition. In the first situation, for three of the 19 mutants, the mutant endosperm was less extreme (the embryo helped); for seven cases, the mutant endosperm was more extreme (the embryo hindered); and for nine cases, there was no change. In the reverse situation, for four cases the normal endosperm helped the mutant embryo; for 14 cases there was no change and one case was inconclusive.

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5'-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 10(8)/g sediment close to the sediment surface to less than 10(5)/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5-1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40-121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 10(4)/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5′-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 108/g sediment close to the sediment surface to less than 105/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5–1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40–121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 104/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere. PMID:22203820

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from γ- or fast neutron-irradiated seed. In two separate Dm1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci. PMID:8088530

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

BACKGROUND Selective immunoglobulin A (IgA) deficiency is the most common inherited immunodeficiency disorder worldwide. An early diagnosis is advocated because of the increased risk of infections, autoimmune diseases, and allergic reactions. We investigated the usefulness of a rapid point-of-care test in detecting for IgAdeficiency in a population with a previously unknown prevalence. MATERIAL AND METHODS Altogether, 1000 children aged 11-13 years from randomly selected Lithuanian schools were enrolled. A point-of-care test with a fingertip sample was used to screen for the presence of IgAdeficiency in children whose parents gave consent. Those with suspected IgAdeficiency were referred to hospital for further clinical examination and confirmation of the diagnosis. In addition, their medical histories were compared with those of 30 age- and sex-matched healthy controls. RESULTS IgAdeficiency was suspected in one girl and in three boys on the basis of the rapid test, and the diagnosis was confirmed for all four cases (prevalence 0.4%, 95% confidence interval 0.16-1.02%). There was no difference in disease history or complications between IgA-deficient children and healthy controls. CONCLUSIONS The rapid antibody test is a practical and accurate method to diagnose selective IgAdeficiency in children. The prevalence of IgAdeficiency among Lithuanian schoolchildren is 1:250.

Background Selective immunoglobulin A (IgA) deficiency is the most common inherited immunodeficiency disorder world-wide. An early diagnosis is advocated because of the increased risk of infections, autoimmune diseases, and allergic reactions. We investigated the usefulness of a rapid point-of-care test in detecting for IgAdeficiency in a population with a previously unknown prevalence. Material/Methods Altogether, 1000 children aged 11–13 years from randomly selected Lithuanian schools were enrolled. A point-of-care test with a fingertip sample was used to screen for the presence of IgAdeficiency in children whose parents gave consent. Those with suspected IgAdeficiency were referred to hospital for further clinical examination and confirmation of the diagnosis. In addition, their medical histories were compared with those of 30 age- and sex-matched healthy controls. Results IgAdeficiency was suspected in one girl and in three boys on the basis of the rapid test, and the diagnosis was confirmed for all four cases (prevalence 0.4%, 95% confidence interval 0.16–1.02%). There was no difference in disease history or complications between IgA-deficient children and healthy controls. Conclusions The rapid antibody test is a practical and accurate method to diagnose selective IgAdeficiency in children. The prevalence of IgAdeficiency among Lithuanian schoolchildren is 1:250. PMID:27920422

Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development.

This study aimed to report the results of a novel surgical technique for the reconstruction of adeficient patella during revision total knee arthroplasty (TKA). Twenty-eight patients (30 knees) with adeficient patella were treated with an onlay-type prosthesis and bone-augmenting procedure, using acrylic bone cement and transcortical wiring. The technique was indicated when the thickness of remnant patella was less than 8mm with variable amounts of the peripheral rim. Mean follow-up period was 36.6months (range, 24 to 55months).The respective mean Knee Society scores for knee and function improved from 34.2 and 23 points, preoperatively to 73.5 and 61 points, at final follow-up. One patient experienced patellar fracture 1week after surgery. There were no complications associated with implanted hardware.

Vitamin Adeficiency (VAD) is an important nutritional problem in India, resulting in an increased risk of severe morbidity and mortality. Periodic, high-dose vitamin A supplementation is the WHO-recommended method to prevent VAD, since a single dose can compensate for reduced dietary intake or increased need over a period of several months. However, in India only 34 percent of targeted children currently receive the two doses per year, and new strategies are urgently needed. Recent advancements in biotechnology permit alternative strategies for increasing the vitamin A content of common foods. Mustard (Brassica juncea), which is consumed widely in the form of oil by VAD populations, can be genetically modified to express high levels of beta-carotene, a precursor to vitamin A. Using estimates for consumption, we compare predicted costs and benefits of genetically modified (GM) fortification of mustard seed with high-dose vitamin A supplementation and industrial fortification of mustard oil during processing to alleviate VAD by calculating the avertable health burden in terms of disability-adjusted life years (DALY). We found that all three interventions potentially avert significant numbers of DALYs and deaths. Expanding vitamin A supplementation to all areas was the least costly intervention, at $23-$50 per DALY averted and $1,000-$6,100 per death averted, though cost-effectiveness varied with prevailing health subcenter coverage. GM fortification could avert 5 million-6 million more DALYs and 8,000-46,000 more deaths, mainly because it would benefit the entire population and not just children. However, the costs associated with GM fortification were nearly five times those of supplementation. Industrial fortification was dominated by both GM fortification and supplementation. The cost-effectiveness ratio of each intervention decreased with the prevalence of VAD and was sensitive to the efficacy rate of averted mortality. Although supplementation is the least

Background Vitamin Adeficiency (VAD) is an important nutritional problem in India, resulting in an increased risk of severe morbidity and mortality. Periodic, high-dose vitamin A supplementation is the WHO-recommended method to prevent VAD, since a single dose can compensate for reduced dietary intake or increased need over a period of several months. However, in India only 34 percent of targeted children currently receive the two doses per year, and new strategies are urgently needed. Methodology Recent advancements in biotechnology permit alternative strategies for increasing the vitamin A content of common foods. Mustard (Brassica juncea), which is consumed widely in the form of oil by VAD populations, can be genetically modified to express high levels of beta-carotene, a precursor to vitamin A. Using estimates for consumption, we compare predicted costs and benefits of genetically modified (GM) fortification of mustard seed with high-dose vitamin A supplementation and industrial fortification of mustard oil during processing to alleviate VAD by calculating the avertable health burden in terms of disability-adjusted life years (DALY). Principal Findings We found that all three interventions potentially avert significant numbers of DALYs and deaths. Expanding vitamin A supplementation to all areas was the least costly intervention, at $23–$50 per DALY averted and $1,000–$6,100 per death averted, though cost-effectiveness varied with prevailing health subcenter coverage. GM fortification could avert 5 million–6 million more DALYs and 8,000–46,000 more deaths, mainly because it would benefit the entire population and not just children. However, the costs associated with GM fortification were nearly five times those of supplementation. Industrial fortification was dominated by both GM fortification and supplementation. The cost-effectiveness ratio of each intervention decreased with the prevalence of VAD and was sensitive to the efficacy rate of averted

Infection is an important cause of morbidity throughout childhood. Poor micronutrient status is a risk factor for infection-related morbidity in young children, but it is not clear whether these associations persist during school-age years. We examined the relation between blood concentrations of micronutrient status biomarkers and risk of gastrointestinal and respiratory morbidity in a prospective study of 2774 children aged 5-12 y from public schools in Bogotá, Colombia. Retinol, zinc, ferritin, mean corpuscular volume, hemoglobin, erythrocyte folate, and vitamin B-12 concentrations were measured in blood at enrollment into the cohort. Children were followed for 1 academic year for incidence of morbidity, including diarrhea with vomiting, cough with fever, earache or ear discharge with fever, and doctor visits. Compared with adequate vitamin A status (≥30.0 μg/dL), vitamin Adeficiency (<10.0 μg/dL) was associated with increased risk of diarrhea with vomiting [unadjusted incidence rate ratio (IRR): 2.17; 95% CI: 0.95, 4.96; P-trend = 0.03] and cough with fever (unadjusted IRR: 2.36; 95% CI: 1.30, 4.31; P-trend = 0.05). After adjustment for several sociodemographic characteristics and hemoglobin concentrations, every 10 μg/dL plasma retinol was associated with 18% fewer days of diarrhea with vomiting (P < 0.001), 10% fewer days of cough with fever (P < 0.001), and 6% fewer doctor visits (P = 0.01). Every 1 g/dL of hemoglobin was related to 17% fewer days with ear infection symptoms (P < 0.001) and 5% fewer doctor visits (P = 0.009) after controlling for sociodemographic factors and retinol concentrations. Zinc, ferritin, mean corpuscular volume, erythrocyte folate, and vitamin B-12 status were not associated with morbidity or doctor visits. Vitamin A and hemoglobin concentrations were inversely related to rates of morbidity in school-age children. Whether vitamin A supplementation reduces the risk or severity of infection in children over 5 y of age needs to

Angelman syndrome (AS) is a childhood-onset neurogenetic disorder characterized by functionally severe developmental delay with mental retardation, deficits in expressive language, ataxia, appendicular action tremors and unique behaviors such as inappropriate laughter and stimulus-sensitive hyperexcitibility. Most cases of AS are caused by mutations which disrupt expression of maternal UBE3A. Although some progress has been made in understanding hippocampal-related memory and learning aspects of the disorder using Ube3adeficient mice, the numerous motoric abnormalities associated with AS (ataxia, action tremor, dysarthria, dysphagia, sialorrhea and excessive chewing/mouthing behaviors) have not been fully explored with mouse models. Here we use a novel quantifiable analysis of fluid consumption and licking behavior along with a battery of motor tests to examine cerebellar and other motor system defects in Ube3adeficient mice. Mice with a maternally inherited Ube3adeficiency (Ube3a(m-/p+)) show defects in fluid consumption behavior which are different from Ube3a(m-/p-) mice. The rhythm of fluid licking and number of licks per visit were significantly different among the three groups (m-/p-, m-/p+, m+/p+) and indicate that not only was fluid consumption dependent on Ube3a expression in the cerebellum, but may also depend on low levels of Ube3a expression in other brain regions. Additional neurological testing revealed defects in both Ube3a(m-/p+) and Ube3a(m-/p-) mice in rope climbing, grip strength, gait and a raised-beam task. Long-term observation of fluid consumption behavior is the first phenotype reported that differentiates between mice with a maternal loss of function versus complete loss of Ube3a in the brain. The neuronal and molecular mechanisms underlying mouse fluid consumption defects specifically associated with maternally inherited Ube3adeficiency may reveal important new insights into the pathobiology of AS in humans.

Mutations in the gene for alpha-galactosidase A result in Fabry disease, a rare, X-linked lysosomal storage disorder characterized by a loss of alpha-galactosidase A enzymatic activity. The resultant accumulation of glycosphingolipids throughout the body leads to widespread vasculopathy with particular detriment to the kidneys, heart and nervous system. Disruption in the autophagy-lysosome pathway has been documented previously in Fabry disease but its relative contribution to nervous system pathology in Fabry disease is unknown. Using an experimental mouse model of Fabry disease, alpha-galactosidase Adeficiency, we examined brain pathology in 20-24 month old mice with particular emphasis on the autophagy-lysosome pathway. Alpha-galactosidase A-deficient mouse brains exhibited enhanced punctate perinuclear immunoreactivity for the autophagy marker microtubule-associated protein light-chain 3 (LC3) in the parenchyma of several brain regions, as well as enhanced parenchymal and vascular immunoreactivity for lysosome-associated membrane protein-1 (LAMP-1). Ultrastructural analysis revealed endothelial cell inclusions with electron densities and a pronounced accumulation of electron-dense lipopigment. The pons of alpha-galactosidase A-deficient mice in particular exhibited a striking neuropathological phenotype, including the presence of large, swollen axonal spheroids indicating axonal degeneration, in addition to large interstitial aggregates positive for phosphorylated alpha-synuclein that co-localized with the axonal spheroids. Double-label immunofluorescence revealed co-localization of phosphorylated alpha-synuclein aggregates with ubiquitin and LC3. Together these findings indicate widespread neuropathology and focused axonal neurodegeneration in alpha-galactosidase A-deficient mouse brain in association with disruption of the autophagy-lysosome pathway, and provide the basis for future mechanistic assessment of the contribution of the autophagy-lysosome pathway

The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.

The authors have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis. Images PMID:2332402

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed. PMID:26618142

Langasites have been studied extensively in past for their functional properties and use in telecommunication. A thorough understanding of their ground state is limited by the difficulty in synthesizing new members belonging to this series due to the formation of competing phases such as the garnets. Three magnetic langasites A3Ga3Ge2BeO14 (A=Pr, Nd, and Sm) and a non-magnetic lattice standard La3Ga3Ge2BeO14 were synthesized using the ceramic method. These were further characterized by X-ray diffraction, magnetization, magnetic susceptibility and heat capacity measurements. All three langasites exhibit net antiferromagnetic interactions at low temperatures and no evidence of long range magnetic ordering was observed down to 0.350 K.

For almost 45 years, the experience of Jewish children who were hidden during World War II was considered to be of little importance, particularly with respect to what had taken place in the concentration camps. Their very history was ignored in the many accounts of the Holocaust. It was only at the end of the 1980s that their experience began to be thought of as potentially traumatic. In this paper, the authors report on their psychoanalytical research project concerning the psychological outcomes of those experiences that had remained concealed for such an extraordinarily long latency period. The results are based on the analysis of 60 accounts and on psychoanalytically-oriented group work. The authors show that the trauma experienced by those hidden children was triggered by the retroactive effect of a deferred action [après-coup].

Each plant species in nature harbors endophytes, a community of microbes living within host plants without causing any disease symptom. However, the exploitation of endophyte-based phytoprotectants is hampered by the paucity of mechanistic understandings of endophyte-plant interaction. We here reported two endophytic Streptomyces isolates IFB-A02 and IFB-A03 recovered from a stress-tolerant dicotyledonous plant Artemisia annua L. After the determination of their non-pathogenicity at the genomic level and from the toxin (thaxtomin A, TXT) level, the endophytism of both isolates was supported by their successful colonization in planta. Of the two endophytes, IFB-A03 was further studied for the mechanism of endophyte-conferred phytoprotection owing to its plant growth promotion in model eudicot Arabidopsis thaliana. Using the endophyte-Arabidopsis co-cultivation system into which pathogenic Streptomyces scabies was introduced, we demonstrated that IFB-A03 pre-inoculation could activate the salicylic acid (SA)-mediated plant defense responses upon pathogen challenge. Moreover, IFB-A03 was shown to partially rescue the defense deficiency in eds5 (enhanced disease susceptibility 5) Arabidopsis mutants, putatively acting at the upstream of SA accumulation in the defense signaling pathway associated with the systemic acquired resistance (SAR). These data suggest that endophytic Streptomyces sp. IFB-A03 could be a promising candidate for biocontrol agents against S. scabies--a causative pathogen of common scab diseases prevailing in agronomic systems.

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

The rates of stunting, iron-deficiency anemia, and vitamin Adeficiency in Peru are among the highest in South America. There is little scaled-up experience on how to solve these problems countrywide. To evaluate the Good Start in Life Program during the period from 2000 to 2004. Data on weight, height, hemoglobin, serum retinol, urinary iodine, and age were obtained from children under 3 years of age during two transverse surveys in 2000 and 2004. In 2004, the program covered 75,000 children, 35,000 mothers, and 1 million inhabitants from 223 poor communities. The rate of stunting decreased from 54.1% to 36.9%, the rate of iron-deficiency anemia decreased from 76.0% to 52.3%, and the rate of vitamin Adeficiency decreased from 30.4% to 5.3% (p < .01). The annual cost per child was US$116.50. Adaptations of this participative program could contribute to decreased stunting, iron-deficiency anemia, and vitamin Adeficiency at the national scale in Peru and many other countries.

The absolute binding free energies and binding enthalpies of twelve host-guest systems in the SAMPL5 blind challenge were computed using our attach-pull-release (APR) approach. This method has previously shown good correlations between experimental and calculated binding data in retrospective studies of cucurbit[7]uril (CB7) and β-cyclodextrin (βCD) systems. In the present work, the computed binding free energies for host octa acid (OA or OAH) and tetra-endo-methyl octa-acid (TEMOA or OAMe) with guests are in good agreement with prospective experimental data, with a coefficient of determination (R2) of 0.8 and root-mean-squared error of 1.7 kcal/mol using the TIP3P water model. The binding enthalpy calculations achieve moderate correlations, with R2 of 0.5 and RMSE of 2.5 kcal/mol, for TIP3P water. Calculations using the newly developed OPC water model also show good performance. Furthermore, the present calculations semi-quantitatively capture the experimental trend of enthalpy-entropy compensation observed, and successfully predict guests with the strongest and weakest binding affinity. The most populated binding poses of all twelve systems, based on clustering analysis of 750 ns molecular dynamics (MD) trajectories, were extracted and analyzed. Computational methods using MD simulations and explicit solvent models in a rigorous statistical thermodynamic framework, like APR, can generate reasonable predictions of binding thermodynamics. Especially with continuing improvement in simulation force fields, such methods hold the promise of making substantial contributions to hit identification and lead optimization in the drug discovery process.

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

Klebsiella pneumoniae mutants were obtained after UV irradiation and negative selection with anticapsular serum. Unencapsulation, rather than expression of a structurally altered capsule, was found in the mutants. The mutant strains showed no alterations in their outer membrane proteins and lipopolysaccharide, and a great similarity with the wild type in the properties tested (serum resistance, antimicrobial sensitivity, and lipopolysaccharide-specific bacteriophage sensitivity), with the exception of a higher cell surface hydrophobicity and resistance to bacteriophage FC3-9.

3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several time-points, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, while others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, class 1 mutants show midline separation defects, corresponding to epithelial junction defects; class 2 mutants show reduced brain ventricle size; class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. PMID:19051268

Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

Resistance of a seedling to the herbicide 1,1′-ethylene-2,2′-dipyridylium bromide (diquat) can be used as a selective technique for photosynthesis mutants in Zea mays L. Diquat requires reduction by the light reaction in order to kill leaf cells and, therefore, nonphotosynthetic mutants survive. This technique was tested using known mutants and is applicable to larger samples of plants than previous techniques. Resistance to diquat should allow selection of mutants on the oxidizing side of photosystem II which are not previously available in higher plants. Images PMID:16659467

Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.

Selenate-resistant mutants were obtained from several strains of Schizosaccharomyces pombe. The obtained mutants all belonged to the same genetic complementation group. They were low in sulphate uptake activity and in ATP sulphurylase activity. They grew on medium containing sulphite, thiosulphate, cysteine or glutathione but not methionine as the sole source of sulphur. From these results, the mutants were concluded to carry mutations in the ATP sulphurylase gene. Inability of the mutants to utilize methionine as a sulphur source is rationalized by the absence of the reverse transsulphurylation pathway in this organism; wild type strains must utilize methionine as a sulphur source after it is degraded to give rise to sulphate.

The prevalence of vitamin Adeficiency (serum retinol [SR] < 20 microg/dl) in children from one to five years of age in the Philippines rose from 35.8% to 38% between 1993 and 1998, despite a twice-yearly universal vitamin A capsule distribution program. The Philippines 1998 National Nutrition Survey, with one-time SR measurements from 11,620 children from one to four years of age, collected over an eight-month period from one month to more than six months after distribution of vitamin A capsules, was an opportunity to examine the impact of the program on the children's vitamin A status, using post hoc analysis. Overall, a detectable impact of vitamin A capsules on SR was limited to groups with the highest prevalence of vitamin Adeficiency and lasted up to four months after dose administration. In highly urban cities in Visayas, where very high prevalences of deficient SR (SR < 10 microg/dl) were found, the prevalence of deficient SR was reduced from 27% to 9% one to two months after distribution of vitamin A capsules, and to 16% at three to four months. In Mindanao, a statistically significant reduction from 38% to 32% was seen in the prevalence of deficient to low SR (SR < 20 microg/dl) one to four months after distribution of vitamin A capsules. There was no overall reduction in the prevalence of vitamin Adeficiency or deficient and low SR (SR < 20 microg/dl) in Luzon, but a significant interaction with stunting was observed in Luzon non-highly urbanized cities. Two aspects are of concern. First, the magnitude of the effect of high-dose vitamin A capsules on SR, and hence on the extent of reduction in deficiency, is limited. Second, the effect does not persist for six months, which is the interval between doses. Thus there is no decrease in the prevalence of deficiency over time. With more frequent dosing (especially to those most deficient in SR), a progressive reduction in vitamin Adeficiency could, however, be expected; this hypothesis could be tested. The

Methods are described for the isolation and testing of temperature-sensitive plasmodial strains of Physarum polycephalum. Nineteen temperature-sensitive strains were found by screening plasmodia derived from mutagenised amoebae and the properties of these are described. A scheme is outlined for the detection of specific mitotic cycle lesions amongst temperature-sensitive strains, and the properties of a presumptive mitotic cycle mutant are described.

Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG

Here, we report a structural analysis of three human amyloid-beta binding alcohol dehydrogenase (ABAD) mutations, identified in patients with 2-methyl-3-hydroxylbutyryl-coA dehydrogenase (MHBD) deficiency. Mapping of the mutations (R130C, L122V, and N247S) on ABAD crystal structure revealed that they occur in the interfaces of the enzyme tetramer. The wild-type and mutant enzymes were then subjected to molecular dynamics simulations with the intention of studying the local effects of the mutations on protein structure. A computational alanine scanning mutagenesis study has been carried out to study the possible impact of the mutations in the energetic contribution of the mutation sites to the binding free energy of ABAD subunit association. In this study, the MMPB-SA (molecular mechanics/Poisson-Boltzmann surface area) approach has been used to calculate the free energy differences on alanine mutation. The interactions and conservation of the mutation sites have been also evaluated. Our results provide an explanation for the strong effect of the R130C mutation on protein stability, evidenced from experimental results. Possibly, the primary effect of this mutation is to impair dimer assembly, as it changes the hot spot character of position 130 to null spot and causes the loss of important hydrogen bonds mediated by the R130 side chain, including a conserved interface hydrogen bond. The other two mutations do not significantly change the energetic contribution of residues 122 and 247 to subunit association, but they are predicted to cause structural changes that affect the enzymatic activity.

Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.

To analyze the status of vitamin Adeficiency among children under 5 years old in poor rural areas of 6 Western Chinese provinces in 2009. The study subjects involved 2091 children from a program - 'Appropriate measurements in improving children under-nutrition in poor rural areas'. In 2009, prevalence of vitamin Adeficiency among children under 5 years old from the poor rural areas of 6 western Chinese provinces was 20.2%, with boys as 20.6% and girls as 19.7%. Prevalence rates of vitamin Adeficiency among children under 5 years old in the highest and lowest areas were seen in Gansu (25.5%) and Guangxi (12.2%) provinces, respectively. The peak of vitamin Adeficiency prevalence fell in the 6 - 11 months age group. The prevalence of vitamin Adeficiency declined along with the increase of age. from this study demonstrated that vitamin Adeficiency among children under 5 years old appeared to be a serious public health problem in the poor rural areas of 6 Western Chinese provinces. Education on reasonable ways of feeding and provision of complementary nutrients to children with vitamin Adeficiency, should be the key points in the prevention and control programs on this problem.

Summary. The RAD6, RAD50, and RAD52 loci have been identified as genes which code for functions which may act during meiotic recombination in yeast (Game et al.1980; Prakash et al. 1980). By use of the spol3-1 mutation,which allows sporulating cells to bypass the first meiotic division, the rad50-1 mutation has been directly implicated as a general meiotic Rec- mutation by examination of viable ascospores (Malone and Esposito 1981). Since the rad6-1 and rad52-1 mutations do not yield viable ascospores in the presence of spol3-1, multiple rad mutants have been constructed and analyzed. This analysis has demonstrated that in meiosis tad50-1 is epistatic to rad52-1, and rad6-1 is epistatic to rad50-1. This suggests that the order of action of these genes during meiosis is RAD6, RAD50, and then RAD52. The data for rad6-1 can be interpreted to suggest that RAD6 may not code for a recombination function,per se, although it may be required for recombination to occur. Analysis of mitotic recombination indicates that rad52-1 is epistatic to rad50-1 ; in mitosis; this is consistent with the hypothesis that the RAD50 gene codes for a recombination function required in meiosis but not in mitosis.

A strain (DA82) having peculiar osmotic properties was isolated in Salmonella typhimurium. The mutant shows increased elasticity of its cell wall and makes spherical instead of elongated cells, regardless of the osmolality of the medium. The strain withstands dilution in distilled water without disruption or death and grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies exponentially in low-osmolality broth (40 milliosmol). Addition of salts or sucrose instantly stops death and allows growth and cell division to proceed. Death is not due to lysis because this appears at later times and at a much lower rate. Osmotic inactivation is temperature-dependent: higher death rates occur at higher incubation temperatures. Inhibition of protein synthesis by chloramphenicol (20 μg/ml) prevents osmotic death. At 37 C and at lower temperatures, the phenomenon of osmotic death is transient. After a variable interval, growth of the osmotic-sensitive strain resumes. It is assumed that the strain's osmotic behavior is due to membrane defectiveness. The membrane disfunction and the wall defect shown by the strain may be consequences of a single genetic alteration or the results of independent mutations. Images PMID:4110928

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation. Images PMID:6434514

A strain (DA82) having peculiar osmotic properties was isolated in Salmonella typhimurium. The mutant shows increased elasticity of its cell wall and makes spherical instead of elongated cells, regardless of the osmolality of the medium. The strain withstands dilution in distilled water without disruption or death and grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies exponentially in low-osmolality broth (40 milliosmol). Addition of salts or sucrose instantly stops death and allows growth and cell division to proceed. Death is not due to lysis because this appears at later times and at a much lower rate. Osmotic inactivation is temperature-dependent: higher death rates occur at higher incubation temperatures. Inhibition of protein synthesis by chloramphenicol (20 mug/ml) prevents osmotic death. At 37 C and at lower temperatures, the phenomenon of osmotic death is transient. After a variable interval, growth of the osmotic-sensitive strain resumes. It is assumed that the strain's osmotic behavior is due to membrane defectiveness. The membrane disfunction and the wall defect shown by the strain may be consequences of a single genetic alteration or the results of independent mutations.

Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in adeficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis. PMID:4622900

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape.

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants. PMID:3470286

A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. PMID:26358292

Dunaliella salina (Chlorophyta, Chlorophyceae) is a unicellular wall-less biflagellate alga. In this paper we describe a spontaneous mutant of D. salina, isolated from wild type cultures, which is characterized by very short flagella. The ultrastructure showed the basic 9 + 2 organization of wild-type flagella. Immunofluorescence localization of tubulin in this mutant confirmed the normal construction of the axoneme. Although, the mutant does not swim, still it is able to move and perform photobehavior. As shown by track reconstruction, and rotation movements, observed by means of reflection microscopy, this mutant can move, probably gliding by means of its stumpy flagella. A possible model to explain the mutant motion pattern is discussed.

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro473 to Leu and Gly737 to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly737 → Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro473 → Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro473 → Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro473 → Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs. PMID:18723844

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype. PMID:24466322

Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI-a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X-linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI-adeficient isolates, real-time analysis for all the 25 autosomal GPI-a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5’ region of PIGL (17p12-p22) extending 5’ (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI-adeficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI-a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously. PMID:25970100

Tay-Sachs disease (TSD) results from mutations in the HEXA gene that cause Hex Adeficiency. Enzyme screening for disease prevention has been applied in the Ashkenazi Jewish and French Canadian populations which have an elevated disease incidence. However, benign mutations that cause Hex Adeficiency, but not TSD, complicate enzyme screening programs. While benign mutations account for only about 2% of Jewish carriers, they account for about 36% of non-Jewish enzyme-defined carriers. We have found a carrier frequency of 1/72 (n=1300) among persons of French Canadian background living in New England using an enzyme-based assay. The HEXA gene of these carriers and others was analyzed to determine the molecular basis of Hex Adeficiency in this group. DNA samples were tested for common previously identified mutations; samples in which no change was found were screened for uncommon or novel mutations using SSCP analysis. Exons showing mobility shifts were sequenced and most mutations were confirmed by restriction enzyme digestion. Known disease-causing mutations were found in 8 samples (4 had a 7.6 kb deletion found in 80% of French Canadian TSD alleles) and known benign mutations were found in 4 samples. Seven novel mutations (G748A; +18 IVS-10 G-to-A; T1338C; +94 IVS-14 T-to-G; C1164G; +30 IVS-6 T-to-G) were identified; the G748A (Gly250Ser) change was found in 3 samples. The effects of the novel mutations on Hex A is unknown; some are likely polymorphisms. The molecular basis of this carrier population is clearly different from that of French Canadian TSD patients. Screening centers should be aware of the presence of benign mutations in the French Canadian population. Given the frequency of the Gly250Ser mutation, and the fact that it has been detected in a TSD patient, it too may be benign.

Kinetics of serum levels of interleukin-6 (IL-6) were studied in patients with acute Plasmodium falciparum malaria in relation to vitamin A and its binding proteins, retinol binding protein (RBP) and pre-albumin. It was found that IL-6 levels followed the rise and decrease of parasitaemia by 12 hr and correlated inversely with levels of vitamin A and its binding proteins. These data suggest that vitamin A supplementation alone might still be insufficient to restore a malaria-induced vitamin Adeficiency. PMID:1572702

The authors study the effect of experimental vitamin Adeficiency on absorption of macromolecules of hen's ovalbumin in the intestine. An electron-microscopic study of permeability of small intestine enterocytes for particles of colloidal lanthanum hydroxide La(OH)/sub 3/ was carried out at the same time. The concentration of unsplit hen's ovalbumin in the blood of the rats used in the experiment was determined by competitive radioimmunoassay. Samples of serum were incubated with indicator doses of /sup 125/I-OA. Radioactivity of the precipitates was measured.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders with multifactorial origin characterized by social communication deficits and the presence of repetitive behaviors/interests. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we assessed the behavioral, neurochemical, and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in the ultrasonic vocal repertoire and a general delay in motor development of reeler pups. We now report that adult male heterozygous (Het) reeler mice did not show social behavior and communication deficits during male-female social interactions. Wildtype and Het mice showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection) only Het mice showed an over response to stress. In addition to the behavioral studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in Het mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD neurobehavioral phenotype.

Two respiratory-deficient nuclear petites, FY23 delta pet191 and FY23 delta cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23 rho(0). Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant, Ki, of 2.3% (w/v) and a specific rate of ethanol production, qp, of 0.90 g ethanol g dry cells-1 h-1. FY23 rho(0) was the most sensitive to ethanol, exhibiting a Ki of 1.71% (w/v) and qp of 0.87 ethanol g dry cells-1 h-1. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23 delta pet191, having a Ki of 2.14% (w/v) and the 85% respiratory-deficient FY23 delta cox5a, having a Ki of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23 rho(0) is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation.

SsrA is a small RNA playing a crucial role in trans-translation, which leads to rescue of stalled ribosomes on or at the end of mRNA and addition of the degradation tag to a growing polypeptide. The lack of SsrA has been shown to enhance the temperature-sensitive (ts) phenotype of an E. coli strain defective in the degP gene, which encodes one of the periplasmic proteases. This severe ts phenotype was relieved only partially by an SsrADD variant, which can lead to ribosome rescue but adds a protease-resistant tag instead of the degradation tag, suggesting that accumulation of polypeptides programmed by truncated mRNAs is responsible for growth defect of the ssrA degP mutant. Expression of an S210A-mutant DegP protein, which lacks the protease activity but retains the chaperone activity, could relieve the ts phenotype of the double mutant, suggesting that the chaperone activity but not the protease activity of DegP is required for growth of the ssrA-deficient cells at high temperature. Overexpression of the rpoE gene, which encodes sigmaE responsible for the expression of factors involved in extracellular stress response, also suppressed the ts phenotype of the ssrA degP mutant. This suggests that the stress-responsing pathway(s) may be involved in the enhancement of ts phenotype of degP mutant in the absence of SsrA.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing. PMID:9055412

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transducin (G{sub t}), was investigated by flash photolysis in order to monitor directly G{sub t} binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of G{sub t}. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border to the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind G{sub t}, mutants CD2 and EF1 showed normal G{sub t} binding but failed to release G{sub t} in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound G{sub t}.

We present a comprehensive, computational study of the properties of bacteriophage λ mutants designed by Atsumi and Little (2006 Proc. Natl. Acad. Sci. 103 4558-63). These phages underwent a genetic reconstruction where Cro was replaced by a dimeric form of the Lac repressor. To clarify the theoretical characteristics of these mutants, we built a detailed thermodynamic model. The mutants all have a different genetic wiring than the wild-type λ. One group lacks regulation of PRM by the lytic protein. These mutants only exhibit the lysogenic equilibrium, with no transiently active PR. The other group lacks the negative feedback from CI. In this group, we identify a handful of bi-stable mutants, although the majority only exhibit the lysogenic equilibrium. The experimental identification of functional phages differs from our predictions. From a theoretical perspective, there is no reason why only 4 out of 900 mutants should be functional. The differences between theory and experiment can be explained in two ways. Either, the view of the λ phage as a bi-stable system needs to be revised, or the mutants have in fact not undergone a modular replacement, as intended by Atsumi and Little, but constitute instead a wider systemic change.